TW201829467A - Anti-CD3 antibody and molecule containing the same - Google Patents

Abstract

The present invention provides a novel antibody that binds to human CD3, or a molecule that has antigen-binding properties comprising the antibody.

Provision of a novel antibody that binds to human CD3, a molecule that has antigen-binding properties of the antibody, and a pharmaceutical composition that contains the antibody or the molecule as an active ingredient and has cytotoxic activity.

Description

   Anti-CD3 antibody and molecule containing the same   

The present invention relates to a novel antibody that binds to human CD3 and to cynomolgus monkey CD3, and a molecule containing the antibody.

1) The anti-CD3 monoclonal antibody system functions like other arbitrary monoclonal antibodies by highly recognizing their target molecules. The anti-CD3 antibody system recognizes only a single epitope on the target CD3 molecule. The most widely used specific monoclonal antibody in the CD3 complex and the best feature is OKT3.

2) OKT3 is a mouse-derived anti-human CD3 monoclonal antibody (Non-Patent Document 1). In order to prevent the rejection of allografts of the organ, an anti-CD3 monoclonal antibody is administered. The antibody binds to the TCR complex on human T cells. By inhibiting the activation and proliferation of T cells and preventing the rejection of transplanted organs, Used for a long time (Non-Patent Documents 2-4). OKT3 is the first anti-CD3 antibody used for this type of treatment. OKT3 has a strong immunosuppressive effect. On the other hand, its clinical use is blocked due to serious side effects related to its immunogenicity and potential ability to promote division (Non-Patent Documents 5-8).

3) OKT3 induces T cell proliferation and cytokine production in vitro, releases a large amount of cytokine in vivo, and causes cytokine syndrome (Non-Patent Document 5). Because OKT3 is a bivalent IgG, it cross-links T cells and Fcγ receptor-expressing cells, resulting in T cell proliferation (Non-Patent Document 8). Since OKT3 is a mouse antibody, it is known to produce a heterophilic antibody such as a human anti-mouse antibody (HAMA) through long-term administration (Non-Patent Document 7). The reports on the use of anti-CD3 antibodies for treatment and side effects are summarized below (Patent Document 1).

4) In order to solve such problems, scFv of OKT3 (Non-Patent Document 9), or humanized OKT3 (Non-Patent Document 10), etc. have been produced. As a further application example of OKT3, a bispecific antibody that binds a single chain of OKT3 that utilizes T cell activation energy to a single chain of an antibody against a target antigen expressed on the surface of cancer cells has been reported (Patent Document 2, Non-Patent Document 11).

5) Multispecific antibodies using anti-CD3 antibodies described in the current technology area are expected to have great therapeutic potential for the treatment of malignant diseases. For example, it is known that TROP2 is overexpressed in various types of epithelial cancer (Non-Patent Documents 12-16). No bispecific antibody has been reported that the antigen-binding fragment of a human TROP2-specific antibody and the antigen-binding fragment of an anti-CD3 antibody are genetically linked.

6) Although OKT3 reacts with CD3 of chimpanzees, it does not react with CD3 derived from other primates such as cynomolgus monkeys (Non-Patent Document 17). UCHT-1, which is an anti-CD3 monoclonal antibody, also reacts with CD3 derived from chimpanzees, but does not react with CD3 derived from cynomolgus monkeys (Non-Patent Document 18). On the other hand, examples of monoclonal antibodies that recognize cynomolgus monkey antigens but have not identified their human counterparts are also seen. One example of this group is FN-18 directed to a monoclonal antibody derived from CD3 of cynomolgus monkey (Non-Patent Document 19).

7) The limitation of a series of antibodies that modify OKT3 and OKT3 is that it only specifically binds to human CD3. Such limitations can be a significant obstacle in the development of therapeutics for the treatment of human diseases. This is because in order to develop candidate medicines and gain market recognition, it is necessary to implement preclinical trials. In preclinical trials, it is expected to use animals, especially higher primate cynomolgus monkeys for testing. Therefore, in the case of using a candidate drug of an anti-CD3 antibody, it is highly desirable to use an anti-CD3 antibody that can bind to CD3 of both humans and cynomolgus monkeys.

8) Antibodies that bind to both humans and cynomolgus monkeys have been reported in Patent Documents 3 and 4, and Non-Patent Document 20. Such a single chain of an anti-CD3 antibody and a bispecific antibody that binds to a single chain of an antibody against a target antigen expressed on the surface of cancer cells have also been reported (Patent Document 5, Non-Patent Document 21). However, in order to be able to use multispecific antibodies and multispecific molecules that are likely to adapt to diverse cancer targets, anti-CD3 antibodies that bind to the different epitopes described above and to both humans and cynomolgus monkeys are expected.

Prior art literature Patent literature

Patent Document 1 International Publication No. 2012/162067

Patent Document 2 International Publication No. 2007/108152

Patent Document 3 US Patent Publication No. 8236308

Patent Document 4 International Publication No. 2008/119567

Patent Document 5 International Publication No. 2015/026892

Non-patent literature

Non-Patent Document 1 Salmeron A. et al., J. Immunol. (1991) 147, 3047-3052

Non-Patent Document 2 Cosmi AB. Et al., Transplantation (1981) 32, 535-539

Non-Patent Document 3 Gilbert EM. Et al., Am. J. Med. (1987) 82, 202-206

Non-Patent Document 4 Thistlethwaite JR. Et al., Transplanation (1987) 43, 176-184

Non-Patent Document 5 Abramowicz D. et al., Transplanation (1989) 47, 606-608

Non-Patent Document 6 Toussaint D. et al., Transplanation (1989) 48, 524-526

Non-Patent Document 7 Thistlethwaite, JR. Et al., Am. J. Kidney Dis. (1988) 11, 112-119

Non-Patent Document 8 Meuer, SC. Et al., Eur. J. Immunol. (1986) 136, 4106-4112

Non-Patent Document 9 George AJ. Et al., J. Immunol. (1994) 152 (4), 1802-11

Non-Patent Document 10 Woodle ES. Et al., J Immunol. (1992) 148 (9), 2756-63

Non-Patent Document 11 Yankelevich M. et al., Pediatr. Blood Cancer (2012) 59 (7), 1198-1205

Non-Patent Document 12 Qhmachi T. et al., Clin. Cancer Res. (2006) 12 (18), 3857-3863

Non-Patent Document 13 Muhlmann G., et al., J. Clin. Pathol. (2009) 62 (2), 152-158

Non-Patent Literature 14 Fong D., et al., Br. J. Cancer (2000) 99 (8), 1290-1295.

Non-Patent Document 15 Fong D. et al., Mod. Pathol. (2000) 21 (2) (2000), 186-191

Non-Patent Document 16 Ning S., et al., Neurol. Sci. (2013) 34 (10), 1745-1750

Non-Patent Document 17 Sandusky et al., J. Med. Primatol. (1986) 15, 441-451

Non-Patent Literature 18 http://www.nhpreagents.org/NHP/clonelist.aspx? ID = 77

Non-Patent Literature 19 Uda et al., J. Med. Primatol. (2001) 30, 141-147

Non-Patent Document 20 Conrad ML. Et al., Cytometry A. (2007) 71 (11), 925-33

Non-Patent Document 21 Lum LG. Et al., BioDrugs (2011) 25 (6), 365-379.

Summary of invention

An object of the present invention is to provide a novel antibody that binds to human CD3 and cynomolgus CD3, or an antigen-binding fragment of the antibody (hereinafter, also referred to as an antibody, etc.), contains the antibody, etc., and one or two or more other The antibody or the molecule of the antigen-binding fragment of the antibody, the molecule having multiple specificities, the pharmaceutical composition containing the antibody or the like or the molecule as an active ingredient and having cytotoxic activity.

The present inventors have conducted intensive research in order to solve the title problem, and created a novel anti-CD3 antibody and a molecule containing the same, and completed the present invention.

That is, the present invention includes the following inventions.

(1) An antibody or an antigen-binding fragment of the antibody, characterized in that the heavy chain sequence includes: CDRH1 including the amino acid sequence shown in sequence identification number 26, and amino acid shown in sequence identification number 98; CDRH2 of the sequence, and CDRH3 including the amino acid sequence shown in SEQ ID NO: 28; the light chain sequence includes: CDRL1 including the amino acid sequence shown in SEQ ID NO: 29, and amine shown in sequence ID 99 CDRL2 of the amino acid sequence and CDRL3 including the amino acid sequence shown in SEQ ID NO: 31; and bind to human CD3 and cynomolgus monkey CD3.

(2) The antibody or the antigen-binding fragment of the antibody according to the above (1), characterized in that the first X _aa of the above-mentioned CDRH2 is selected from (A, E, G, H, I, L, T, V , R, S), and the second X _aa is S, or the first X _aa is N, and the second X _aa is selected from (E, R, F, Y, L, V , I, K, T), X _aa of the aforementioned CDRL2 is selected from the group consisting of (Q, A, G, S, N, D), and binds to human CD3 and cynomolgus CD3 .

(3) The antibody or the antigen-binding fragment of the antibody according to (1) or (2), wherein the first X _aa of the aforementioned CDRH2 is selected from the group consisting of (R, S), and Two X _aa are S, and the X _aa of the aforementioned CDRL2 is selected from the group consisting of (Q, A, G, S, N, D), and binds to human CD3 and cynomolgus monkey CD3.

(4) The antibody or the antigen-binding fragment of the antibody according to (1) above, characterized in that the heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, and the CDRH1 is represented by SEQ ID NO: 26 The amino acid sequence is composed of the amino acid sequence shown in SEQ ID No. 27, and the above-mentioned CDRH3 is composed of the amino acid sequence shown in SEQ ID NO: 28; and the light chain sequence includes CDRL1. , CDRL2, and CDRL3 variable regions. The aforementioned CDRL1 is composed of the amino acid sequence shown in SEQ ID NO: 29, the aforementioned CDRL2 is composed of the amino acid sequence shown in SEQ ID NO: 30, and the aforementioned CDRL3 is composed of a sequence It consists of the amino acid sequence shown by the identification number 31, and binds to human CD3 and cynomolgus CD3.

(5) The antibody or the antigen-binding fragment of the antibody according to any one of (1) to (4), wherein the heavy chain variable region sequence includes an amino acid sequence represented by sequence identification number 100.

(6) The antibody or antigen-binding fragment thereof according to (5) above, wherein the first X _aa of the amino acid sequence represented by the sequence identification number 100 is selected from (A, E, G, H, I, L, T, V, R, S), and the second X _aa is S, or the first X _aa is N, and the second X _aa is selected from (E, R, F, Y, L, V, I, K, T).

(7) The antibody or antigen-binding fragment thereof according to (5) above, wherein the first X _aa of the amino acid sequence shown by sequence identification number 100 is selected from the group consisting of (R, S), and The second X _aa is S.

(8) The antibody or antigen-binding fragment thereof according to any one of (1) to (7) above, characterized in that the variable region of the light chain includes any one of sequence identification numbers 101, 102, and 103. Amino acid sequence.

(9) The antibody or antigen-binding fragment thereof according to (8) above, wherein X _aa of the amino acid sequence shown by any one of sequence identification numbers 101, 102, and 103 is selected from (Q, A, G, S, N, D).

(10) The antibody or the antigen-binding fragment of the antibody according to the above (5), characterized in that the heavy chain variable region sequence includes an amino acid sequence represented by sequence identification number 16.

(11) The antibody or antigen-binding fragment thereof according to (8) above, wherein the light chain variable region sequence includes an amino acid sequence shown in any one of sequence identification numbers 17, 20, and 23 .

(12) The antibody or antigen-binding fragment thereof according to (1) or (2), comprising: a heavy chain variable region comprising an amino acid sequence represented by sequence identification number 100; and a sequence identifier 101, The light chain variable region of the amino acid sequence shown in any of 102, and 103, and the first X _aa of the amino acid sequence shown in sequence identification number 100 is selected from (A, E, G, H , I, L, T, V, R, S), and the second X _aa is S, or the first X _aa is N, and the second X _aa is selected from (E, R , F, Y, L, V, I, K, T), X _aa of the amino acid sequence shown in any one of the sequence identification numbers 101, 102, and 103 is selected from (Q, A, G, S, N, D).

(13) The antibody or antigen-binding fragment thereof according to (12) above, wherein the first X _aa of sequence identification number 100 is selected from the group consisting of (R, S), and the second X _aa is S, In addition, X _aa of the amino acid sequence shown by any one of the sequence identification numbers 101, 102, and 103 is selected from the group consisting of (Q, A, G, S, N, D).

(14) The antibody or the antigen-binding fragment thereof according to the above (13), which comprises a heavy chain variable region containing an amino acid residue containing sequence identification number 2 to 119 and sequence recognition Antibodies to the light chain variable region of amino acid residues of Nos. 60 to 135 to 243 or antigen-binding fragments of the antibodies; heavy chain variable containing amino acid residues containing sequence identification numbers 64 to 119 Region, and an antibody or an antigen-binding fragment of the light chain variable region comprising amino acid residues of 135 to 241 of sequence identification number 64; an amino acid residue of 2 to 119 including sequence identification number 66 The heavy chain variable region of the amino acid group, and the antibody or the antigen-binding fragment thereof comprising the light chain variable region of the amino acid residues of amino acid residues 135 to 243 of sequence identification number 66; The heavy chain variable region of the amino acid residue of 119 and the antibody or the antigen-binding fragment of the antibody comprising the light chain variable region of the amino acid residue of sequence number 68 to 135 to 243; Heavy chain variable regions of amino acid residues 2 to 119 of the identification numbers 70 and 135 to 243 Antibodies to light chain variable regions of amino acid residues or antigen-binding fragments of the antibodies; heavy chain variable regions containing amino acid residues containing sequence identification numbers 72 to 119, and sequence identification number 72 Antibody of the light chain variable region of an amino acid residue of 135 to 243 or an antigen-binding fragment of the antibody; a heavy chain variable region comprising an amino acid residue of SEQ ID NO: 74 2 to 119, And an antibody or an antigen-binding fragment thereof comprising a light chain variable region of an amino acid residue of SEQ ID NO: 74 to 135 to 243; and an antibody comprising an amino acid residue of SEQ ID NO: 76 to 2 to 119 Antibodies or antigen-binding fragments of heavy chain variable regions and light chain variable regions comprising amino acid residues of amino acid residues 135 to 243 of sequence identification number 76; An antibody or an antigen-binding fragment of the heavy chain variable region of an amino acid residue and the light chain variable region of an amino acid residue of SEQ ID NO: 78 to 135 to 243; Heavy chain variable regions of amino acid residues 2 to 119 of 80, and 135 to 243 including sequence identification number 80 Antibodies to light chain variable regions of amino acid residues or antigen-binding fragments of the antibodies; heavy chain variable regions containing amino acid residues containing sequence identification numbers 82 to 119, and sequence identification numbers An antibody to the light chain variable region of an amino acid residue of 135 to 243 of 82 or an antigen-binding fragment of the antibody; or a heavy chain variable comprising an amino acid residue of SEQ ID NO: 84 to 119 Region, and an antibody or an antigen-binding fragment of the antibody comprising a light chain variable region of amino acid residues of sequence identification numbers 84 to 243.

(15) The antibody or the antigen-binding fragment of the antibody according to the above (1), (4), (5), (8), (10), or (11), which contains the sequence identification number 16 A heavy chain variable region of the amino acid sequence, a linker, and a light chain variable region including the amino acid sequence shown in any one of sequence identification numbers 17, 20, and 23.

(16) The antibody or antigen-binding fragment thereof according to any one of (1) to (15), wherein the variable region is from the amine terminal side of the antibody, and the heavy chain variable region and the light chain variable region are Or in the order of the light chain variable region and the heavy chain variable region, and optionally: i) have a linker between the two variable regions; ii) one of the variable regions on the amine terminal side The amino-terminus has a glycine residue; iii) a linker, a FLAG tag, and / or a His-tag are bonded to the carboxy-terminus of the variable region on the carboxy-terminus side.

(17) The antigen or the antigen-binding fragment of the antibody according to the above (16), comprising: an amino acid sequence including an amino acid residue at positions 2 to 243 of sequence identification number 60; and a sequence identification number Amino acid sequence of amino acid residues at positions 2 to 241 of 64, amino acid sequence including amino acid residues at positions 2 to 243 of sequence identification number 66, second sequence including sequence identification number 68 The amino acid sequence of the amino acid residues from positions 243 to 243, the amino acid sequence of the amino acid residues from positions 2 to 243 of sequence identification number 70, the amino acid sequence of positions 2 to 243 including the sequence identification number 72 Amino acid residues of amino acid residues, amino acid residues including amino acid residues at positions 2 to 243 of sequence identification number 74, amino acid residues including amino acid residues of positions 2 to 243 of sequence identification number 76 Amino acid sequence of amino group, amino acid sequence including amino acid residues at positions 2 to 243 of sequence identification number 78, amino acid sequence including amino acid residues of positions 2 to 243 of sequence identification number 80 Acid sequence, an amino acid sequence comprising an amino acid residue at positions 2 to 243 of sequence identification number 82, or an amino acid residue comprising an amino acid residue at positions 2 to 243 of sequence identification number 84 The amino acid sequence.

(18) The antigen or the antigen-binding fragment of the antibody according to the above (16), comprising: an amino acid sequence including amino acid residues of sequence identification number 2 to 269; and an amino acid sequence including sequence identification number 22 Amino acid sequences of amino acid residues 2 to 269, amino acid sequences including amino acid residues 2 to 267 of sequence identification number 25, amino acid residues including 2 to 269 of sequence identification number 60 Amino acid sequence including amino acid residues of 2 to 267 of sequence identification number 64, amino acid sequence including amino acid residues of 2 to 269 of sequence identification number 66, including Amino acid sequences of amino acid residues 2 to 269 of sequence identification number 68, amino acid sequences containing amino acid residues of 2 to 269 of sequence identification number 70, 2 to 269 including sequence identification number 72 Amino acid residues of the amino acid residues of the amino acid residues, amino acid sequences of the amino acid residues of sequence identification numbers 74 2 to 269, amines containing the amino acid residues of the sequence identification numbers 2 of 2 to 269 Amino acid sequence, amino acid residues containing amino acid residues 2 to 269 of sequence identification number 78, amino acid residues containing amino acid residues of 2 to 269 of sequence identification number 80 Amino acid sequence comprising the amino acid sequence of SEQ ID NO amino acids 2-269 of 82 residues, or the amino acid sequence comprising SEQ ID NO 84 the amino acid residues of 2-269.

(19) An antibody or an antigen-binding fragment of the antibody, comprising an amino acid sequence and binding to human CD3 and cynomolgus monkey CD3, wherein the amino acid sequence is encoded by a nucleotide sequence and the nuclear The nucleotide sequence is contained in a polynucleotide that hybridizes to a complementary strand of the following polynucleotide under stringent conditions: encoding the antibody as described in any one of the above (14) to (18) or an antigen-binding fragment of the antibody Polynucleotide sequence containing a nucleotide sequence.

(20) An antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain includes binding to the antibody according to any one of the above (14) to (18) or an antigen of the antibody The amino acid sequence of the heavy chain contained in the sex fragment is 90% or more of the amino acid sequence, and the light chain contains the antibody described in any one of the above (14) to (18) or an antigen of the antibody The amino acid sequence of the light chain contained in the binding fragment is more than 70% identical; and it binds to human CD3 and cynomolgus monkey CD3.

(21) An antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises: the antibody or the antibody comprising the antibody according to any one of (14) to (18) above; Among the amino acid sequences of the heavy chain contained in the antigen-binding fragment, one to several amino acids are substituted, deleted, or added; and the light chain includes: (14) to (18) The amino acid sequence of the light chain contained in the antibody or the antigen-binding fragment of the antibody, wherein one to several amino acids are substituted, deleted, or added Amino acid sequence; and bind to human CD3 and cynomolgus monkey CD3.

(22) an antibody or an antigen-binding fragment of the antibody, which binds to the same site on human CD3 to which the antibody or the antigen-binding fragment of the antibody according to any one of (14) to (18) above, And bind to cynomolgus monkey CD3.

(23) An antibody or an antigen-binding fragment of the antibody, which competes with the antibody or the antigen-binding fragment of the antibody described in any one of (14) to (18) above for binding to human CD3, and Crab-eating macaque CD3 binding.

(24) The antibody or the antigen-binding fragment of the antibody according to (22), wherein the site on human CD3 to which the antibody or the antigen-binding fragment of the antibody binds is an amine group represented by sequence identification number 1 In the acid sequence, serine (Ser) at position 55, glutamic acid (Glu) at position 56, leucine (Leu) at position 58, tryptophan (Trp) at position 59, Aspartic acid (Asn) at position 66, Ileucine at position 66 (Ser), Serine at position 77 (Asp), Asp at position 101 Arginine (Arg), Glycine (Gly) at position 101, Serine (Ser) at position 103, Lysine (Lys) at position 104, and Proline (Pro at position 105) ) Is composed of 7 or more amino acids.

(25) The antibody or the antigen-binding fragment of the antibody according to any one of (1 to 17), (19) to (24) above, which is an IgG.

(26) The antibody or the antigen-binding fragment thereof according to any one of (1) to (23), which is selected from the group consisting of Fab, F (ab) ', Fv, scFv, and sdAb.

(27) The antibody or the antigen-binding fragment of the antibody according to any one of (1) to (17), (19) to (25) above, which is a humanized antibody comprising a human immunoglobulin constant region or Human antibodies.

(28) A polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of the antibody or the antigen-binding fragment of the antibody according to any one of the above (1) to (27).

(29) The polynucleotide according to the above (27), comprising a nucleotide sequence encoding the following amino acid sequence: an amino acid sequence including an amino acid residue at positions 2 to 243 of sequence identification number 19 , An amino acid sequence comprising an amino acid residue at positions 2 to 243 of sequence identification number 22, an amino acid sequence comprising an amino acid residue at positions 2 to 241 of sequence identification number 25, including sequence identification The amino acid sequence of amino acid residues at positions 2 to 243 of number 60, the amino acid sequence of amino acid residues at positions 2 to 241 of sequence identification number 64, and the amino acid sequence of sequence number 66 Amino acid sequences of amino acid residues 2 to 243, amino acid sequences including amino acid residues 2 to 243 of sequence identification number 68, and 2 to 243 amino acid sequences including sequence identification number 70 Amino acid residues of amino acid residues, amino acid residues including amino acid residues at positions 2 to 243 of sequence identification number 72, amino acid residues including amino acid residues of positions 2 to 243 of sequence identification number 74 Amino acid sequence of residues, amino acid sequence including amino acid residues 2 to 243 of sequence identification number 76, amino acid sequence including 2 to 243 of sequence identification number 78 Amino acid residues containing amino acid residues, amino acid residues containing amino acid residues at positions 2 to 243 of sequence identification number 80, amino acid residues including amino acid residues at positions 2 to 243 of sequence identification number 82 Or an amino acid sequence comprising an amino acid residue at positions 2 to 243 of sequence identification number 84.

(30) A vector comprising the polynucleotide according to any one of (28) or (29) above.

(31) A cell comprising the polynucleotide according to any one of (28) or (29) above or the vector according to (30) above, or producing any one of (1) to (27) above The antibody or the antigen-binding fragment thereof according to the item.

(32) A method for producing an antibody that binds to human CD3 and cynomolgus monkey CD3 or an antigen-binding fragment of the antibody, comprising the steps of culturing the cell according to the above (31); and recovering from the culture and human A step of a CD3-bound antibody or an antigen-binding fragment of the antibody.

(33) An antibody that binds to human CD3 and cynomolgus CD3, or an antigen-binding fragment of the antibody, which is obtained by the method described in (32) above.

(34) A pharmaceutical composition comprising the antibody according to any one of (1) to (27) and (33) or an antigen-binding fragment of the antibody as an active ingredient.

(35) An antigen-binding molecule comprising the antibody according to any one of (1) to (27) and (33) or an antigen-binding fragment of the antibody.

(36) The molecule according to (35) above, which is multispecific.

(37) The molecule according to the above (35) or (36), comprising the antibody according to any one of the above (1) to (27) and (33) or an antigen-binding fragment of the antibody, and one Or two or more additional antibodies or antigen-binding fragments of the antibody.

(38) The molecule according to (37) above, wherein the antigen-binding fragment of another antibody is Fab, F (ab) ', Fv, scFv, or sdAb.

(39) The molecule according to (38) above, which comprises Fc.

(40) The molecule according to any one of (37) to (39), wherein the additional antibody is a humanized antibody or a human antibody comprising a human immunoglobulin constant region.

(41) The molecule according to any one of the aforementioned (37) to (38), wherein the additional antibody or an antigen-binding fragment of the antibody is the same as any of the aforementioned (1) to (27), (33) The antibody or antigen-binding fragment of one item described above is formed by binding to a linker or not.

(42) The molecule according to the above (41), wherein the carboxyl terminal of the amino acid sequence of the additional antibody or the antigen-binding fragment of the antibody is bound to a linker, and the amino acid possessed by the linker is further The carboxyl terminus of the sequence is bound to the antibody or the antigen-binding fragment of the antibody according to any one of (1) to (27) and (33) above.

(43) The molecule according to the above (42), comprising: the carboxyl terminus of the amino acid sequence of the additional antibody or the antigen-binding fragment of the antibody is bound to a linker, and further includes the linker Amino acid sequence in which the carboxy terminus of the amino acid sequence is combined with the following antibody or an antigen-binding fragment of the antibody: an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 19 or an antibody An antigen-binding fragment, an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 22 or an antigen-binding fragment of the antibody, or an amino acid residue comprising positions 2 to 241 of sequence identification number 25 Based antibody or an antigen-binding fragment of the antibody.

(44) The molecule according to any one of the above (35) to (42), which is an antibody according to any one of the above (1) to (27) and (33) or an antigen-binding fragment of the antibody In the present invention, the variable region is bound from the amine terminal side of the antibody in the order of the heavy chain variable region and the light chain variable region, or in the order of the light chain variable region and the heavy chain variable region, optionally: i) a linker between the two variable regions; ii) an amino terminal of the variable region on the amine terminal side with a glycine residue; iii) a carboxy terminal of the variable region on the carboxy terminal side, binding There are linkers, FLAG tags, and / or His tags. Applicable forms include bispecific molecules of hybrid type (hybrid type) and dual type (dual type).

(45) The molecule according to the above (44), which comprises the additional antibody or an antigen-binding fragment of the antibody, and an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 19 or the antibody An antigen-binding fragment thereof, an antibody comprising an amino acid residue at positions 2 to 241 of SEQ ID NO: 25, or an antigen-binding fragment of the antibody.

An antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 60 or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 241 of sequence identification number 64, or the antibody An antigen-binding fragment thereof, an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 66 or an antigen-binding fragment of the antibody, an amino acid residue comprising an amino acid residue at positions 2 to 243 of sequence identification number 68 Antibody or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 70, or an antigen-binding fragment of the antibody, comprising 2 to 243 of SEQ ID NO: 72 Antibody having an amino acid residue at the position 1, or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 74, or an antigen-binding fragment of the antibody, including a sequence identification number An antibody to an amino acid residue at positions 2 to 243 of 76 or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 78, or the antigen-binding property of the antibody Fragment containing amino acid residues 2 to 243 of sequence identification number 80 The antibody or an antigen-binding fragment thereof, an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 82 or an antigen-binding fragment of the antibody, or SEQ ID NO: 84 from 2 to 243 An amino acid residue of the antibody or an antigen-binding fragment of the antibody. In the applicable form, it includes hybrid and dual type bispecific molecules.

(46) The molecule according to any one of (36) to (45), wherein the additional antibody is an anti-cancer target antibody.

(47) The molecule according to any one of (36) to (46) above, which is bispecific.

(48) The molecule according to any one of (35) to (47) above, which is a polypeptide.

(49) A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence possessed by the molecule according to the above (48).

(50) A vector comprising the polynucleotide according to the above (49).

(51) A cell producing a polynucleotide according to the above (49) or a vector according to the above (50) or a molecule according to the (48).

(52) A method for producing a molecule that binds to human CD3 and cynomolgus CD3, comprising a step of culturing the cell according to the above (51), and a step of recovering the molecule that binds to human CD3 from the culture.

(53) A molecule binding to human CD3 and cynomolgus monkey CD3, which is obtained by the method described in (52) above.

(54) A pharmaceutical composition containing the molecule according to any one of (35) to (48) and (53) as an active ingredient.

(55) The pharmaceutical composition according to the above (54), wherein a T cell is used to redirect a target cell to induce cytotoxicity to the target cell.

According to the present invention, a novel anti-CD3 antibody that binds to human CD3 and cynomolgus monkey CD3, or an antigen-binding fragment of the antibody, and a novel molecule that includes the antibody and has antigen-binding properties can be obtained. In addition, a novel pharmaceutical composition containing such an antibody or the like as an active ingredient can be obtained. The antibody or the like has a T-cell-dependent cytotoxic activity and is useful as a therapeutic or preventive agent for various diseases such as cancer.

FIG. 1 is a diagram showing the amino acid sequence of human CD3ε.

Figure 2 is a diagram showing the amino acid sequence of human CD3δ.

FIG. 3 is a diagram showing a nucleotide sequence encoding a human CD3εγ single-chain antigen.

FIG. 4 is a diagram showing the amino acid sequence of a human CD3εγ single chain antigen.

FIG. 5 is a diagram showing the nucleotide sequence of a sense primer Nhe-polyC-S for heavy chain gene amplification.

FIG. 6 is a diagram showing the nucleotide sequence of the first anti-sense primer rIgγ-AS1 for heavy chain gene amplification.

FIG. 7 is a diagram showing the nucleotide sequence of the second antisense primer rIgγ-AS2 for heavy chain gene amplification.

Fig. 8 is a diagram showing the nucleotide sequence of a sense primer Nhe-polyC-S2 for light chain gene amplification.

FIG. 9 is a diagram showing the nucleotide sequence of the first antisense primer rIgL-AS1 for light chain gene amplification.

FIG. 10 is a diagram showing the nucleotide sequence of the second antisense primer rIgL-AS2 for light chain gene amplification.

FIG. 11 is a diagram showing a nucleotide sequence of a sense primer rIgγ-seq for heavy chain sequencing.

FIG. 12 is a diagram showing the nucleotide sequence of the antisense primer 1rIgL-seq1 for light chain sequencing.

FIG. 13 is a diagram showing the nucleotide sequence of the antisense primer 2rIgL-seq2 for light chain sequencing.

FIG. 14 is a diagram showing a nucleotide sequence encoding a heavy chain variable region of C3-147.

Figure 15 is a diagram showing the amino acid sequence of the heavy chain variable region of C3-147.

FIG. 16 is a diagram showing a nucleotide sequence encoding a light chain variable region of C3-147.

Figure 17 is a diagram showing the amino acid sequence of the light chain variable region of C3-147.

Fig. 18 is a diagram showing the oligonucleotide sequence of the sense strand of the G4S linker.

FIG. 19 is a diagram showing the oligonucleotide sequence of the antisense strand of the G4S linker.

FIG. 20 is a diagram showing a nucleotide sequence encoding C3E-7000.

Figure 21 is a diagram showing the amino acid sequence of C3E-7000.

Figure 22 is a diagram showing the amino acid sequence of the heavy chain variable region of C3E-7034.

Figure 23 is a diagram showing the amino acid sequence of the light chain variable region of C3E-7034.

Figure 24 is a diagram showing the amino acid sequence of CDR-H1 of C3E-7000.

Figure 25 is a diagram showing the amino acid sequence of CDR-H2 of C3E-7000.

Figure 26 is a diagram showing the amino acid sequence of CDR-H3 of C3E-7000.

Figure 27 is a diagram showing the amino acid sequence of CDR-L1 of C3E-7000.

Figure 28 is a diagram showing the amino acid sequence of CDR-L2 of C3E-7000.

Figure 29 is a diagram showing the amino acid sequence of CDR-L3 of C3E-7000.

Figure 30 is a diagram showing the amino acid sequence of the light chain variable region of C3E-7035.

Figure 31 is a diagram showing the amino acid sequence of C3E-7035.

Figure 32 is a diagram showing the amino acid sequence of the light chain variable region of C3E-7036.

Figure 33 is a diagram showing the amino acid sequence of C3E-7036.

Figure 34 is a diagram showing a nucleotide sequence encoding C3E-7034.

FIG. 35 is a diagram showing a nucleotide sequence encoding C3E-7035.

Figure 36 is a diagram showing a nucleotide sequence encoding C3E-7036.

Figure 37 is a diagram showing the amino acid sequence of human CD3γ.

Figure 38 is a diagram showing the amino acid sequence of C3E-7034.

FIG. 39 is a diagram showing a complex structure of CD3εγ and C3E-7034.

FIG. 40 is a diagram showing the interaction of CD3εγ with the heavy and light chains of C3E-7034.

Line A shows the light chain variable region of C3E-7034 and the amino acid residues of CD3ε with CD3ε within 4Å of each other in the thick rod model, and other amino acids in the thin rod model.

B shows the heavy chain variable region of C3E-7034 and the amino acid residues of CD3ε with CD3ε within 4Å of each other in the thick rod model, and other amino acids in the thin rod model.

Figure 41 is a diagram showing the interaction sites on the sequences of the heavy chain variable region and light chain variable region of human CD3ε with C3E-7034.

Figure 42 is a diagram showing the amino acid sequence of the heavy chain variable region of OKT3.

Figure 43 is a diagram showing the amino acid sequence of the heavy chain variable region of C3E-3007.

Figure 44 is a diagram showing the amino acid sequence of the light chain variable region of OKT3.

Figure 45 is a diagram showing the amino acid sequence of the light chain variable region of C3E-3007.

Figure 46 is a diagram showing a nucleotide sequence encoding a C3E-3007 scFv.

Figure 47 is a diagram showing the amino acid sequence of C3E-3007 scFv.

Figure 48 is a diagram showing the binding properties of C3E-3007, C3E-7034, C3E-7035, and C3E-7036 to human CD3 (PBMC) as humanized anti-CD3scFv.

Figure 49 is a graph showing the binding properties of C3E-3007, C3E-7034, C3E-7035, and C3E-7036, which are humanized anti-CD3 scFv, to cynomolgus CD3 (PBMC).

In FIG. 50, line A shows the activation of human CD8-positive cells by C3E-7034 and C3E-3007, which are humanized anti-CD3 scFv. Line B shows the activation of CD8-positive cells of C3E-7034 and C3E-3007 cynomolgus monkeys that are humanized anti-CD3 scFv.

Figure 51 is a diagram showing the amino acid sequence of HT1-11 scFv.

Figure 52 is a diagram showing a nucleotide sequence encoding HT1-11 scFv.

Figure 53 is a diagram showing the binding of HT1-11 scFv to a human TROP2 positive cell line. Line A shows the binding to the pharyngeal squamous epithelial cancer cell line FaDu. Line B shows the binding to the pancreatic cancer cell line HPAF-II.

Figure 54 is a diagram showing the nucleotide sequence of the ORF encoding T2C-0001.

Figure 55 is a diagram showing the ORF nucleotide sequence encoding T2C-0003.

Figure 56 is a diagram showing the ORF nucleotide sequence encoding T2C-0005.

Figure 57 is a diagram showing the nucleotide sequence of the ORF encoding T2C-0006.

Figure 58 is a diagram showing the amino acid sequence of T2C-0001.

Figure 59 is a diagram showing the amino acid sequence of T2C-0003.

Figure 60 is a diagram showing the amino acid sequence of T2C-0005.

Figure 61 is a diagram showing the amino acid sequence of T2C-0006.

FIG. 62 is a diagram showing the binding of the anti-TROP2-CD3 bispecific molecule, T2C-0001, T2C-0003, T2C-0005, and T2C-0006 to a TROP2-positive cell line. Line A shows the binding to the pharyngeal squamous epithelial cancer cell line FaDu. Line B shows the binding to the pancreatic cancer cell line HPAF-II.

Figure 63 is a diagram showing the binding properties of anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006, to human CD3 (PBMC).

FIG. 64 is a diagram showing the binding properties of anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006, to cynomolgus CD3 (PBMC).

Fig. 65

Line A is a graph showing the appearance of TROP2 in a pharyngeal flat epithelial cancer cell line FaDu.

The B line is shown in the pancreatic cancer cell line HPAF-II and shows the appearance of TROP2.

C line is a lung cancer cell line Calu-6, and there is no graph showing TROP2 expression.

Fig. 66

Line A shows anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006 against pharyngeal squamous epithelial cancer cell line FaDu, which has cytotoxic activity in the coexistence of human PBMC.

Line B presents a map of anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006, showing cytotoxic activity against pancreatic cancer cell line HPAF-II in the presence of human PBMC.

Line C showed anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006 against human lung cancer cell line Calu-6. In the coexistence of human PBMC, no cytotoxic activity was shown.

Fig. 67 is a diagram showing a nucleotide sequence encoding C3E-7078.

Figure 68 is a diagram showing the amino acid sequence of C3E-7078.

FIG. 69 is a diagram showing a nucleotide sequence encoding C3E-7079.

Figure 70 is a diagram showing the amino acid sequence of C3E-7079.

FIG. 71 is a diagram showing a nucleotide sequence encoding C3E-7085.

Figure 72 is a diagram showing the amino acid sequence of C3E-7085.

FIG. 73 is a diagram showing a nucleotide sequence encoding C3E-7086.

Figure 74 is a diagram showing the amino acid sequence of C3E-7086.

Figure 75 is a diagram showing a nucleotide sequence encoding C3E-7087.

Figure 76 is a diagram showing the amino acid sequence of C3E-7087.

Figure 77 is a diagram showing a nucleotide sequence encoding C3E-7088.

Figure 78 is a diagram showing the amino acid sequence of C3E-7088.

Figure 79 is a diagram showing a nucleotide sequence encoding C3E-7089.

Figure 80 is a diagram showing the amino acid sequence of C3E-7089.

FIG. 81 is a diagram showing a nucleotide sequence encoding C3E-7090.

Figure 82 is a diagram showing the amino acid sequence of C3E-7090.

Fig. 83 is a diagram showing a nucleotide sequence encoding C3E-7091.

Figure 84 is a diagram showing the amino acid sequence of C3E-7091.

FIG. 85 is a diagram showing a nucleotide sequence encoding C3E-7092.

Figure 86 is a diagram showing the amino acid sequence of C3E-7092.

Fig. 87 is a diagram showing a nucleotide sequence encoding C3E-7093.

Figure 88 is a diagram showing the amino acid sequence of C3E-7093.

Fig. 89 is a diagram showing a nucleotide sequence encoding C3E-7094.

Figure 90 is a diagram showing the amino acid sequence of C3E-7094.

Figure 91 is a diagram showing a nucleotide sequence encoding C3E-7095.

Figure 92 is a diagram showing the amino acid sequence of C3E-7095.

FIG. 93 is a diagram showing a nucleotide sequence encoding a primer HN53R Fw.

Fig. 94 is a diagram showing a nucleotide sequence encoding a primer HN53R Rv.

Fig. 95 is a diagram showing a nucleotide sequence encoding a primer HN53S Fw.

Fig. 96 is a diagram showing a nucleotide sequence encoding a primer HN53S Rv.

Fig. 97 is a diagram showing a nucleotide sequence of an ORF encoding AXC-0001.

Fig. 98 is a diagram showing the amino acid sequence of AXC-0001.

Fig. 99 is a diagram showing a nucleotide sequence of an ORF encoding AXC-0002.

FIG. 100 is a diagram showing the amino acid sequence of AXC-0002.

FIG. 101 is a diagram showing a nucleotide sequence of an ORF encoding MGC-0001.

Figure 102 is a diagram showing the amino acid sequence of MGC-0001.

Fig. 103 is a diagram showing a nucleotide sequence of an ORF encoding MGC-0002.

Figure 104 is a diagram showing the amino acid sequence of MGC-0002.

Figure 105 is a list of primers used to make CDR variants of anti-CD3 antibodies.

FIG. 106-1 is a diagram showing the binding properties of various anti-CD3 antibody CDR variants to human and cynomolgus CD3 (PBMC).

Figure 106-2 is a diagram showing the binding properties of various anti-CD3 antibody CDR variants to human and cynomolgus CD3 (PBMC).

Figure 107 shows human lung cancer cell line A549 (A), human pancreatic cancer cell line PANC-1 (B), human pancreatic cancer cell line MIA PaCa-2 (C), human myeloma cell line U266B1 (D), A graph showing the expression of Axl in a mantle cell lymphoma cell line Jeko-1 (E).

In FIG. 108, A, B, and C lines show anti-Axl-CD3 bispecific molecules, AXC-0001, and AXC-0002, which show Axl-expressing cell lines and have cytotoxic activity in the coexistence of human PBMC. Lines D and E show anti-Axl-CD3 bispecific molecules, AXC-0001, and AXC-0002 on Axl non-expressing cell lines, coexisting with human PBMC, but showing no cytotoxic activity.

FIG. 109 is a diagram showing the binding properties of MAG-032scFv in MAGEC1 lymphoma (A) or DMSO-added (B) human lymphoblast fusion cell line T2 cells.

In FIG. 110, line A shows anti-HLA-A2 / MAGEC1-CD3 bispecific molecules, MGC-0001, and MGC-0002 on T2 cells to which MAGEC1 peptide is added, which have cytotoxic activity in the presence of human PBMC. Line B is a diagram showing the case where the anti-HLA-A2 / MAGEC1-CD3 bispecific molecule, MGC-0001, and MGC-0002 paired with DMSO-added T2 did not show cytotoxic activity in the presence of human PBMC.

Figure 111 is a diagram showing the amino acid sequence of the MAGEC1 peptide.

Figure 112 is a diagram showing the amino acid sequence of the CDRH2 region of the CDR variant. The first X _aa and the second X _aa are each an arbitrary amino acid residue.

FIG. 113 is a diagram showing the amino acid sequence of the CDRL2 region of the CDR variant. X _aa is any natural amino acid residue.

Figure 114 is a diagram showing the heavy chain amino acid sequence of the CDR variant of C3E-7034. The first X _aa and the second X _aa are each an arbitrary amino acid residue.

Figure 115 is a diagram showing the light chain amino acid sequence of the CDR variant of C3E-7034. CDRL2 any of the natural amino acid-based X _aa residues.

Figure 116 is a diagram showing the light chain amino acid sequence of the CDR variant of C3E-7035. CDRL2 any of the natural amino acid-based X _aa residues.

Figure 117 is a diagram showing the light chain amino acid sequence of the CDR variant of C3E-7036. CDRL2 any of the natural amino acid-based X _aa residues.

Forms used to implement the invention     Definition    

In the present invention, "gene" means a nucleotide contained in a base sequence encoding an amino acid of a protein or a complementary strand thereof, for example, a nucleotide contained in a base sequence encoding an amino acid of a protein Polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., or their complementary strands are included in the meaning of "gene". The gene is a single-stranded, double-stranded, or triple-stranded nucleotide. The DNA strand meets the RNA strand. There are ribonucleotides (RNA) and deoxyribonucleotides on the single-stranded nucleotide strand. (DNA) mixed and double-stranded or triple-stranded nucleotides containing such a nucleotide chain are also included in the meaning of "gene". In the present invention, a base sequence has the same meaning as a nucleotide sequence.

In the present invention, "polynucleotide", "nucleic acid" and "nucleic acid molecule" have the same meaning. For example, DNA, RNA, probes, oligonucleotides, and primers are also included in the meaning of "polynucleotide". The polynucleotide comprises a single-stranded, double-stranded or more than three-stranded polynucleotide, a confluence of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribose on a single-stranded polynucleotide strand Mixtures of nucleotides (DNA) and the combination of double or triple-strands containing the polynucleotide are also included in the meaning of "polynucleotide".

In the present invention, "polypeptide", "peptide" and "protein" have the same meaning.

In the present invention, "antigen" is sometimes used in the meaning of "immunogen".

In the present invention, the "cell" also includes various cells derived from animal individuals, subculture cells, primary culture cells, cell lines, recombinant cells, microorganisms, and the like.

In the present invention, "antibody" is synonymous with immunoglobulin. However, the "antibody" in the case of the present invention called an anti-CD3 antibody is used in the sense of an immunoglobulin having a constant region and a variable region. The antibody is a natural one or an immunoglobulin produced by partial or complete synthesis, but it is not particularly limited. The anti-CD3 antibody system of the present invention is included in a "molecule" described later.

The structure of a basic 4-chain antibody is composed of two identical light (L) chains and two identical heavy (H) chains. The light chain is bound to the heavy chain via a covalent disulfide bond. The two heavy chains are bonded to each other by one or a plurality of disulfide bonds in response to the isotype of the heavy chain. The respective light and heavy chains are regularly spaced and have intra-chain disulfide bonds. In the heavy and light chains, there are a constant region in which the amino acid sequence shows a very high similarity and a variable region in which the amino acid sequence has a low similarity. The light chain is attached to the constant region (CL) and has a variable region (VL) at the amino terminal end. The heavy chain is connected to three constant regions (CH1 / CH2 / CH3) and has a variable region (VH) at the amine terminal. VL is paired with VH, and CL is aligned with the first constant region (CH1) of the heavy chain. VL is paired with VH to form a single antigen-binding site.

Although the constant region of the antibody of the present invention is not particularly limited, it is preferable to use a human antibody for the antibody of the present invention for treating or preventing human diseases. Examples of the heavy chain constant region of a human antibody include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε. Examples of the light chain constant region of a human antibody include Cκ and Cλ.

Fab is composed of CH1 of the heavy chain and VH following it, and CL of the light chain and VL following it. The VH and VL lines contain complementarity determining regions (CDRs).

Fc is the carboxy-terminal region of the constant region of the heavy chain, and contains CH2 and CH3, which is a dimer. The Fc of the present invention may be a Fc of a natural sequence, or a Fc of a mutant type in which a mutation is added to the natural sequence.

The variable region is composed of a region having extreme variability called a hypervariable region (HVR) and a relatively constant region called a framework region (FR) separated by the region. The natural heavy chain and light chain variable regions contain 4 FRs followed by 3 hypervariable regions. The hypervariable regions of each chain use FRs to maintain close proximity with the hypervariable regions of the other chain. Contributes to the formation of the antigen-binding site of the antibody.

For the heavy chain and light chain of an antibody molecule, it is known that there are three complementarity determining regions (CDRs). The complementarity-determining region is also called a hypervariable region. It is a site with extremely high primary variability in the variable regions of the heavy and light chains of the antibody. It is located on the primary structure of the heavy and light polypeptide chains. Usually, they are separated in 3 places. In the present invention, regarding the complementarity determining region of an antibody, the amino acid terminal side of the amino acid sequence of the heavy chain self-heavy chain is labeled as CDRH1, CDRH2, CDRH3, and the complementarity determining region of the light chain is determined from the light chain. The amino acid terminal side of the amino acid sequence is labeled CDRL1, CDRL2, CDRL3. These sites are close to each other in the three-dimensional structure, which determines the specificity of the bound antigen.

In the present invention, the position and length of the CDRs are determined by the definition of IMGT (Developmental and Comparative Immunology 27 (2003) 55-77).

The framework region (FR) is a variable region other than the CDR residues. The variable region generally has four FRs of FR1, FR2, FR3, and FR4.

The positions of CDR and FR are various definitions known in the technical area. For example, besides IMGT, they can also be determined according to the definitions of Kabat, Chothia, AbM, contact, etc.

In the present invention, the "antigen-binding fragment of an antibody" means a partial fragment of an antibody having binding activity to an antigen, which is composed of a variable region of a heavy chain and a variable region of a light chain. Examples of the "antigen-binding fragment of an antibody" include antigen-binding fragments such as Fab, F (ab ') ₂ , scFv, Fab', Fv, and a single-domain antibody (sdAb). But it is not limited to them. The antigen-binding fragment of the antibody is obtained by treating the full-length molecule of the antibody protein with enzymes such as papain and pepsin, as well as a recombinant protein produced by using a recombinant gene in an appropriate host cell.

In the present invention, the "site" to which the antibody binds, that is, the "site" recognized by the antibody means a partial peptide or a partial higher order structure on the antigen to which the antibody binds or recognizes.

In the present invention, this site is also referred to as an epitope and an antibody binding site. In the present invention, "antibody variant" means an amino group in which the amino acid sequence of the original antibody has amino acid substitution, deletion, addition (including addition and insertion) (hereinafter, collectively referred to as "variation"). Acid sequence, and a polypeptide that binds to CD3 of the present invention. The number of variant amino acids in the antibody variant is 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50. This antibody variant is also included in the "antibody" of the present invention.

In the present invention, the "number" of "1 to several" means 2 to 10.

In the present specification, the "molecule" refers to a molecule including the above-mentioned antibody or antigen-binding fragment of an antibody, and further includes a multispecific molecule formed by an antibody or a plurality of antigen-binding fragments derived therefrom.

In this specification, "multispecific molecules", "multispecific molecules," and "multispecific molecules" have the same meaning, as long as they are different from multiple epitopes on one molecule, It is not particularly limited as long as it is a molecule capable of binding to different epitopes on two or more molecules. Multispecific molecules also include antibodies containing a heavy chain variable region (VH) and a light chain variable region (VL). Such multispecific molecules include fully-long antibody molecules having two or more different types of heavy and light chains, that is, IgG-type multispecific molecules and antigen-binding fragments having two or more types of VL and VH. The resulting molecule is a molecule derived by combining Fab, Fab ', Fv, scFv, sdAb, etc., including tandem scFv, diabody, single chain double chain antibody, triabody, etc. It is not limited to these. Molecules produced by genetically or chemically linking proteins with antigen-binding properties and no immunoglobulin backbone to other antigen-binding fragments are also included in multispecific molecules.

Examples of the activities and properties of the anti-CD3 antibody of the present invention or the antigen-binding fragment of the antibody or the multispecific molecule of the present invention include biological activity, physicochemical properties, and the like. In other words, various biological activities, binding activity to an antigen or epitope, stability during production or storage, heat stability, and the like can be mentioned.

In the present invention, "hybridization under stringent conditions" is performed at 65 ° C in a solution containing 5 × SSC, followed by 20 minutes at 65 ° C in an aqueous solution containing 2 × SSC-0.1% SDS, and 0.5 × Hybridization was performed at 65 ° C for 20 minutes in an SSC-0.1% SDS aqueous solution and at 65 ° C for 20 minutes in an aqueous solution containing 0.2 × SSC-0.1% SDS, and hybridization was performed under respective washing conditions or equivalent conditions. SSC means an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and n × SSC means n times the concentration of SSC.

In the present invention, "cytotoxic" refers to any way that causes pathological changes to cells, including not only direct trauma, but also cutting off DNA or base dimer formation, cutting off chromosomes, and cells. Damage to the division device, reduction of various enzyme activities, and other damage to all cellular structures or functions.

In the present invention, "cytotoxic activity" means those who cause the aforementioned cytotoxicity.

In the present invention, "antibody-dependent cellular cytotoxicity" refers to "antibody dependent cellular cytotoxicity (ADCC) activity", and also refers to the action activity of NK cells to target cells such as tumor cells through antibodies.

In the present invention, "cytotoxic activity due to T cell redirection" means that the aforementioned cytotoxicity is caused by a multispecific molecule comprising an anti-tumor antigen antibody and an anti-CD3 antibody. That is, an anti-tumor antigen antibody binds to a target tumor cell, and an anti-CD3 antibody binds to a T cell, thereby reducing the distance between the target tumor cell and the T cell, and inducing cytotoxicity by activating the T cell. This molecular system may be contained in a pharmaceutical composition.

    2. Antigen protein CD3 (CD3 complex)    

In the present invention, "CD3" is used in the same sense as CD3 protein.

The CD3 line is a part of a multi-molecule T cell receptor complex and is expressed on T cells. It is a five-type polypeptide of γ chain, δ chain, ε chain, ζ chain, and η chain (molecular weights are 25000-28000, 21000, 20000, 16000, 22000).

The CD3 system used in the present invention can be purified from animal tissues (including body fluids), cells derived from the tissues, or the cell culture, and can be prepared by using isolation, genetic recombination, in vitro translation, chemical synthesis, and the like.

The nucleotide sequence of cDNA encoding human CD3ε was registered in GenBank under the accession number: NM_000733.3. The nucleotide sequence of the cDNA encoding the cynomolgus monkey CD3 was registered in GenBank under the accession number: NM_001283615.1. The amino acid sequence of human CD3ε is described in SEQ ID NO: 1 in the Sequence Listing.

For CD3ε cDNA, for example, a polymerase chain reaction (hereinafter referred to as "PCR") can be performed using a cDNA library of organs expressing CD3ε mRNA as a template, and primers that specifically increase CD3ε cDNA (hereinafter referred to as "PCR") (Saiki, RK , Et al., Science (1988) 239, 487-49).

In addition, the amino acid sequence encoded by CD3 contains a nucleotide sequence of one to several amino acids substituted, deleted, or added, and a nucleotide sequence of a protein having the same biological activity as CD3 is also included in The nucleotide sequence of the CD3 gene. In addition, the amino acid sequence in CD3 includes one or more amino acid sequences in which amino acids are substituted, deleted, or added, and proteins having the same biological activity as CD3 are also included in CD3.

In addition, a polynucleotide that is complementary to a nucleotide sequence encoding a nucleotide sequence encoding CD3ε of a human or cynomolgus monkey is hybridized under stringent conditions, and a polynucleotide encoding a protein having a biological activity equivalent to that of CD3ε Also included in CD3ε cDNA. Furthermore, a splicing variant transcribed from a human or cynomolgus CD3ε locus or a polynucleotide that hybridizes to it under stringent conditions, and a polynucleotide encoding a protein having the same biological activity as CD3ε is also included in CD3ε cDNA.

    3. Anti-CD3 antibodies         (3-1) Classification of antibodies    

The anti-CD3 antibody of the present invention and the antigen-binding fragment of the antibody (hereinafter also referred to as the antibody of the present invention, etc.) may be either a single antibody or a multiple antibody. Examples of the monoclonal antibodies of the present invention include non-human animal-derived antibodies (non-human animal antibodies), human antibodies, chimeric antibodies (also referred to as "chimeric antibodies"), and humanized antibodies.

Examples of non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds. Examples of mammalian-derived antibodies include rodent-derived antibodies such as mouse antibodies and rat antibodies. Examples of the bird-derived antibodies include chicken antibodies. Examples of the anti-human CD3 rat monoclonal antibody include C3-147 (Example 1) -7) of the present invention.

Examples of the chimeric antibody include, but are not limited to, antibodies in which a variable region derived from a non-human animal antibody is combined with a human antibody (human immunoglobulin) constant region.

In the case of humanized antibodies, CDRs in the variable regions of non-human animal antibodies can be transplanted to human antibodies (variable regions of human immunoglobulins). In addition to the CDRs, the framework region sequences of non-human animal antibodies can also be cited. Those who are transplanted to human antibodies, and those in which one or more amino acids derived from non-human animal antibodies are substituted with human-type amino acids, and the like are not limited to them. As for the CDRs in the variable region of the non-human animal antibody, CDRH1 to CDRH3 and the light chain variable region in the heavy chain variable region derived from the rat anti-CD3 antibody C3E-7000 of the present invention can be exemplified. The amino acid sequences of CDRL1 to CDRL3 and their CDRs are those in which one or two amino acids are substituted with other amino acids.

The human antibody is not particularly limited as long as it preferably binds to human CD3, and more preferably binds to human CD3 and cynomolgus monkey CD3. A human antibody or the like that binds to the same site as the humanized antibody of the present invention can also be exemplified. For example, a human antibody that binds to the same site as C3E-7034 can be exemplified.

Preferred antibodies and the like of the present invention bind to human CD3. Furthermore, better antibodies and the like of the present invention have the binding activity to CD3 of cynomolgus monkeys.

If the antibody of the present invention binds to human CD3 and also to CD3 of cynomolgus monkey, it may be an antibody composed of a plurality of different antibody-derived portions. The heavy and / or light chain is exchanged, the full length of the heavy and / or light chain is exchanged, only the variable region or constant region is exchanged, all or only a portion of the CDR is exchanged, and so on. The heavy chain variable region and the light chain variable region of a chimeric antibody may be derived from different antibodies of the present invention. CDRH1 to CDRH3 and CDRL1 to CDRL3 in the variable regions of the heavy and light chains of humanized antibodies may be derived from two or more antibodies of the present invention. CDRH1 to CDRH3 and CDRL1 to CDRL3 in the variable regions of the heavy and light chains of a human antibody may be a combination of two or more CDRs possessed by the antibody of the present invention.

The isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgGs such as IgG1, IgG2, IgG3, and IgG4; IgA, IgM, IgA1, and IgA2;

The isotype and subtype of a monoclonal antibody can be determined, for example, by Ouchterlony method, Enzyme-Linked ImmunoSorbent Assay (ELISA) method, Radio Immuoassay (RIA) method, etc. A commercially available identification kit (Rat Immunoglobulin Isotyping ELISA Kit (BD Pharmingen), etc.) can also be used.

    (3-2) Binding specificity of anti-CD3 antibodies    

The antibodies and the like of the present invention recognize CD3. That is, the antibodies and the like of the present invention are bound to CD3. The antibodies and the like of the present invention preferably bind to human CD3, monkey CD3 and the like, and more preferably to human CD3 and cynomolgus CD3.

In more detail, the antibody of the present invention, its antigen-binding fragment and its variable region, and the Ig-like domain (Ig) of the extracellular region existing in the epsilon chain (Figure 1, sequence identification number 1) of the human CD3 complex -like domain). Furthermore, it also binds to Ig-like domains that are present in the extracellular region of the epsilon chain of the cynomolgus CD3 complex.

The epitope in the extracellular region of the ε chain (Figure 1, sequence identification number 1) of the human CD3 complex bound by the antibody of the present invention contains the following amino acids; Ser55, Glu56, Leu58, Trp59, Asn65 , Ile66, Ser77, Asp78, Arg101, Gly102, Ser103, Lys104, and Pro105.

The antibody or the like of the present invention is preferably capable of maintaining binding to human CD3 by binding to an epitope region comprising at least 7 amino acids selected from these 13 amino acids.

When the antibody and the aforementioned amino acid are adjacent to each other within a distance of 4Å, such an antibody can be judged to have the same epitope specificity as the antibody of the present invention. On the other hand, among the above-mentioned epitope amino acids, Arg101, Gly102, Ser103, Lys104, and Pro105 also have epitope residues that interact with known anti-CD3 antibodies OKT3 or UCHT1 (Lars Kjer-Nielsen et al., PNAS (2004) (Kelly L Arnett et al., PNAS (2004)). However, although OKT3 and UCHT1 bind to human CD3, they do not bind to cynomolgus CD3.

In the present invention, "identification", that is, "binding", means not binding non-specifically. As a criterion for determining whether to recognize, that is, whether or not to combine, for example, a dissociation constant (hereinafter referred to as "KD"). The KD value of the preferred antibody of the present invention on CD3 is 1 × 10 ^-5 M or less, 5 × 10 ^-6 M or less, 2 × 10 ^-6 M or less, or 1 × 10 ^-6 M or less.

The binding system of an antigen and an antibody in the present invention can be measured or determined by using an in vivo molecular interaction analysis system such as the SPR method, the BLI method, or the ELISA method, the RIA method, or the like. The binding system of the antigen and antibody expressed on the cell surface can be measured by a flow cytometry method or the like.

The SPR method (Surface Plasmon Resonance analysis method) is used as a solution to determine the binding rate constant (Ka value) and dissociation rate constant (Kd value) by chemical kinetic (kinetic) analysis. An analytical technique such as dissociation constant (KD value), which is an index of affinity, must be obtained. Examples of the machine used for SPR analysis include Biacore (trademark) (made by GE HEALTHCARE), ProteOn (trademark) (made by BioRad), SPR-Navi (trademark) (made by BioNavis), and Spreeta (trademark) ( (Made by Texas Instruments), SPRi-PlexII (trademark) (made by HORIBA), Autolab SPR (trademark) (made by Metrohm), and the like.

The BLI method (BioLayer Interferometry) is a method for measuring the interaction between living molecules using biofilm interference. As an apparatus for interaction analysis using the BLI method, an Octet system (manufactured by Pall ForteBio) can be exemplified.

The ELISA method is a method of detecting and quantifying the target antigen or antibody contained in the sample solution with a specific antibody or antigen while using an enzyme reaction. The enzyme-labeled antigen or antibody is incorporated into the reaction system to detect the enzyme activity. For the detection of enzyme activity, a substrate with a change in the absorption spectrum depending on the reaction is used to quantify the absorbance measurement.

Cell-ELISA is a method of detecting and quantifying each cell by supplementing the measurement target on the cell surface with enzyme reaction.

The RIA method (Radio Immunoassay, Radio Immunoassay) uses radioactive substances to identify antibodies, and by measuring the radioactivity from the antibodies, the antibodies can be quantified.

The flow cytometry method is a method in which fine cells are dispersed in a fluid, the fluid is made to flow, and each cell is optically analyzed. The fluorescein-labeled antibody binds to a cell surface antigen by an antigen-antibody reaction, and the number of cells bound by the antibody is measured using fluorescence intensity to measure the antigen-binding property of the antibody.

As described above, antibodies that bind to human CD3 and cynomolgus monkey CD3 can be better supplied to non-clinical development (preclinical development) of primates that must be used for pharmaceuticals, especially the effectiveness and safety of cynomolgus monkeys. Related tests. In addition, antibodies that bind to human CD3 and cynomolgus CD3 have cytotoxic activity and are used alone or as a molecule of the present invention to treat or prevent diseases such as cancer in humans and cynomolgus monkeys. The pharmaceutical composition is described below.

In addition, because the antibodies binding to human CD3 and cynomolgus CD3 of the present invention do not bind to mouse CD3, they are used in cells, tissues, and individuals (including transgenic animals, knockout animals, and gene knockouts) of mice that have introduced human CD3 genes (Knock in animals) and various analyses, immunohistochemistry, etc. using the antibody can be performed without the influence of CD3 of the host mouse, and the medicine, animal medicine, or diagnostic drug containing the antibody Research and non-clinical development using mice is preferred.

    (3-3) monoclonal antibodies    

The present invention provides a monoclonal antibody. Monoclonal antibodies, including rat antibodies, mouse antibodies, rabbit antibodies, chicken antibodies, fish antibodies, and other non-human animal-derived monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and their antigens Binding fragments, their antibody variants, their modifications, and the like.

For example, C3-147 obtained by the method described in Example 1 is an anti-CD3 rat monoclonal antibody.

The nucleotide sequence of the DNA encoding the heavy chain variable region of C3-147 is described in Sequence ID No. 6 (Figure 14) of the Sequence Listing, and the amino acid sequence is described in Sequence ID No. 7 (Figure 15). The nucleotide sequence of the DNA encoding the light chain variable region of C3-147 is described in Sequence ID No. 8 (Figure 16) of the Sequence Listing, and the amino acid sequence is described in Sequence ID No. 9 (Figure 17).

Preferably, the antibody variant of the present invention can be made to have reduced sensitivity to protein degradation or oxidation, maintenance of biological activity and function, improvement or reduction and inhibition of change, improvement or regulation of antigen binding energy, or physical and chemical properties. Or the imparting of functional properties. It is known that the specific amino acid side chain of a protein is changed on the surface and the function or activity of the protein will be changed. Such examples include deamination of aspartic acid side chain, aspartic acid side chain Isomerization and so on. In order to prevent such changes in the amino acid side chain, those substituted with other amino acids are included in the scope of the antibody variant of the present invention.

Examples of the antibody variant of the present invention include an antibody in which the amino acid sequence of the antibody has an amino acid sequence substituted with a stored amino acid. Preserved amino acid substitutions are those occurring within the amino acid group associated with the amino acid side chain.

Suitable amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basicity group = lysine, arginine, histidine; non-polar group = alanine, valine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine, Tryptophan; and Non-Polarized Family = Glycine, Aspartic Acid, Glutamine, Cysteine Acid, serine, threonine, tyrosine. Other suitable amino groups are as follows: Aliphatic hydroxyl group = serine and threonine; containing amido group = aspartic acid and glutamic acid; aliphatic group = alanine, valine, white Amino acids and isoleucine; and aromatic groups = phenylalanine, tryptophan, and tyrosine. The amino acid substitution in the antibody variant is preferably performed within a range that does not reduce the antigen-binding activity possessed by the original antibody.

The antibody variant having the amino acid sequence of the C3-147-containing antibody (1) or the antigen-binding fragment thereof of the present invention having a stored amino acid substitution and / or other mutations, And any of the amino acid sequences of CDRH1 to CRDH3 and CDRL1 to CDRL3 contained in the above-mentioned (1) antibody or antigen-binding fragment thereof containing C3-147 has a preserved amino acid substitution and / or other modified amino groups A mouse antibody, a rat antibody, a chimeric antibody, a humanized antibody, a human antibody, an antigen-binding fragment thereof, and the like containing the CDRs of the acid sequence are also included in the present invention.

    (3-4) Antigen-binding fragment of anti-CD3 antibody    

An antigen-binding fragment of the anti-CD3 antibody of the present invention is provided as one aspect of the present invention. An antigen-binding fragment of an antibody means a fragment or a modification thereof that maintains at least antigen-binding properties in the function of the antibody. Examples of the function of the antibody include antigen-binding activity, activity for regulating antigen activity, antibody-dependent cytotoxic activity, and complement-dependent cytotoxic activity. Examples of the functions of the antibody and the like of the present invention and the case of including multiple specific molecules of the antibody and the like of the present invention include T cell redirection, activation of T cells, and cancer cells utilizing activated T cells. Cytotoxic activity.

The antigen-binding fragment of the antibody is not particularly limited as long as it has at least antigen-binding fragment of the antibody's activity, and examples include Fab, Fab ', and F (ab') _2. Fv, a single-chain Fv (scFv), a single-domain antibody (sdAb), and the like that link the Fv of a heavy chain and a light chain with an appropriate linker, but are not limited thereto. If the scFv has a linker portion, a molecule containing a portion other than the antigen-binding fragment of the antibody of the present invention is also included in the meaning of the antigen-binding fragment of the antibody of the present invention.

Molecules whose amino acids at the amino and / or carboxyl terminus of an antibody have one to several deletions or more, and which retain at least a part of the functions of the antibody, also include the meaning of the antigen-binding fragments of the antibody . Modifications of the antigen-binding fragments of such antibodies are also included in the antibodies or antigen-binding fragments thereof, or their modifications (described later).

One aspect of the antigen-binding fragment of the antibody of the present invention is scFv. scFv is obtained by linking the heavy and light chain variable regions of an antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, edited by Rosenburg and Moore, Springer Verlag, New York, 269-315 (1994 ), Nature Biotechnology (2005), 23, 1126-1136). Moreover, a tandem scFv prepared by binding two scFvs with a polypeptide linker can also be used as a bispecific molecule. Further, a triple-chain antibody made of three or more scFvs can also be used as a multispecific molecule.

The antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence. This type of antibody is called a single domain antibody (sdAb) or a nanobody, and it has been reported that the antigen-binding ability is maintained (Muyldemans S.et.al., Protein Eng., (1994) 7 (9 ), 1129-35, Hamers-Casterman C. et.al., Nature (1993) 363 (6428), 446-448). These antibodies also include the meaning of the antigen-binding fragments of the antibodies in the present invention.

In the present invention, a single chain immunoglobulin (Lee, HS, et.al., Molecular Immunology (1999) 36 is used to link the full-length sequences of the heavy and light chains of the antibody with an appropriate linker. , 61-71; Shirrmann, T. et.al., mAbs (2010), 2 (1), 1-4). Such a single-chain immunoglobulin may retain a structure and activity similar to that of an antibody that is originally a tetramer by dimerization.

    (3-5) Antigen-binding molecules    

The molecule of the present invention comprises the anti-CD3 antibody of the present invention or an antigen-binding fragment thereof.

The molecule of the present invention may further include the following: a message sequence; a tag for purification and the like; an amino terminal Gly; an ADC drug linker portion; an albumin binding polypeptide; a polymer such as PEG; an antibody other than an anti-CD3 antibody; Its antigen-binding fragments; proteins that do not have immunoglobulin backbone but have antigen-binding properties; compounds (parts) with anti-cancer effects, cytotoxic activities, and other pharmacological activities.

The molecule of the present invention binds to human CD3 and cynomolgus monkey CD3.

The molecule of the present invention includes a multispecific molecule described later.

The molecule of the present invention may be a form introduced into a cell such as CAR-T, or a form presented on a cell surface.

    (3-6) Multispecific molecules, bispecific molecules    

The multispecific molecule of the present invention is a molecule having two or more antigen-binding sites. That is, two or more different epitopes that can bind to one molecule or two different epitopes that can bind to two or more molecules include a plurality of different antigen bindings Sexual fragment. Such multispecific molecules include IgG-type multispecific molecules and multispecific molecules with two or more variable regions, such as tandem scFv, single-chain double-chain antibodies, double-chain antibodies, and triple-chain antibodies. Antibody fragments, antibody fragments that are covalently bonded or non-covalently bonded, but are not limited thereto. The multispecific molecule may comprise an Fc.

The multispecific molecule of the present invention comprises the anti-CD3 antibody of the present invention or an antigen-binding fragment of the antibody. The multispecific molecule of the present invention includes the antibody or the like of the present invention, and one or two or more additional antibodies or antigen-binding fragments of the antibody. Examples of the antigen-binding fragment of another antibody include Fab, F (ab) ', Fv, scFv, and sdAb.

The multispecific molecular system of the present invention can specifically bind to CD3, or can further bind to a target such as an Fc receptor on an effector cell.

Preferred examples of the multispecific molecule of the present invention include bispecific molecules. "Bispecificity" means that it can bind to two epitopes that are different from each other or two epitopes that are different from each other on the same molecule, and includes antibodies or antigen-binding properties with such bispecificity. Fragment. The bispecific molecule of the present invention binds to CD3 and binds to epitopes located on other antigens that do not have CD3. More specifically, the bispecific molecular line (i) binds to an epitope (antigenic position 1) on CD3, and (ii) an epitope different from an epitope 1 on CD3 ( Epitope 2) binds, or binds to an epitope (epitope 3) of an antigen other than CD3.

For example, biTE molecules of the tandem scFv type represented by BiTE, the antigen-binding site of the heavy chain variable region of the first antibody, and the antigen-binding site of the light chain variable region of the first antibody are linked by a linker or not. The linker directly binds to form the first polypeptide, and the antigen-binding site of the heavy chain variable region of the second antibody and the antigen-binding site of the light chain variable region of the second antibody are linked by the linker or not. The linker directly binds to form a second polypeptide, and the first polypeptide and the second polypeptide are directly linked by a linker or not by a linker. In addition, the first polypeptide and the second polypeptide may be bound via another molecule.

In a double-chain antibody-type bispecific molecule, the antigen-binding site of the heavy-chain variable region of the first antibody and the antigen-binding site of the light-chain variable region of the second antibody are linked with or without a linker. In addition, the antigen-binding site of the light chain variable region of the first antibody and the antigen-binding site of the heavy chain variable region of the second antibody are linked with a linker or directly without a linker. It is also possible to produce a bispecific molecule that further dimerizes a double-chain antibody type bispecific molecule. Other double-chain antibody-type bispecific molecules may be linked to one or both of the Fc by a linker (double-chain antibody-Fc-type bispecific molecule).

In a dual scFv (dual scFv) type bispecific molecule, two types of scFvs that bind to different epitopes and one of the dimer Fc are directly linked by a linker or not by a linker. Alternatively, two types of scFvs that bind to different epitopes are linked to CH and CL by linkers, respectively, and one of the Fc of the dimer is linked to the linker. Hereinafter, a dual scFv-type bispecific molecule is also described as a dual-type bispecific molecule or simply as a dual-type.

For IgG-type bispecific molecules, two types of Fabs that bind to different epitopes and one of the Fc of the dimer are linked with a linker or directly without a linker. Hereinafter, bispecific molecules of the IgG type will also be described as full-size antibody (FSA) type bispecific molecules, or will be simply described as the FSA type.

Alternatively, as the bispecific molecule of the present invention, Fabs and scFvs that bind to different epitopes may be bispecific antibodies that are each linked to a dimer Fc with or without a linker. . Or it can be one of the Fc of the dimer, the Fab of the first antibody, and the bispecific molecule of the scFv of the second antibody that has been bound by a linker. Hereinafter, such a bispecific molecule is also described as a hybrid bispecific molecule or a hybrid type.

The scFv and Fab contained in the bispecific molecule of the present invention are preferably scFv and Fab of humanized antibodies or human antibodies, and the Fc is preferably Fc of human antibodies.

The variable region contained in the bispecific molecule of the present invention may be sequentially combined from the heavy chain variable region and the light chain variable region from the amine terminal side of the antibody, or the light chain variable region and the heavy chain may be Sequence of variable zones combined. There is a linker between the two variable regions. The amino group terminal of the variable region on the side of the amino group terminal may have a glycine residue (optionally). In the tandem scFv-type bispecific molecule, the carboxyl terminus of the variable region on the carboxyl terminus side can be combined with a linker, a FLAG tag, and / or a His tag (optionally). In one suitable aspect, from the amine terminal, the heavy chain variable region, the first linker, the light chain variable region, the second linker, the FLAG tag, and the His tag can be exemplified in order. By.

Linkers also include single-chain polypeptides or single-chain oligopeptides, or synthetic products such as PEG, nucleotides, sugar chains, compounds, and the like. Others are not limited as long as they bind two polypeptides, and it is possible to use a known linker.

The length of the linker is, for example, 5 to 30 amino acids in the case of a peptide linker. In the case where the bispecific molecule includes a plurality of linkers, peptide linkers all of the same length may be used, or peptide linkers of different lengths may be used.

As for the peptide linker, for example, a repeat (Gly‧Gly‧Gly‧Gly‧Ser) can be exemplified, and one to several amino acid residues different from Gly and Ser can also be added thereto.

    (3-7) Humanized anti-CD3 antibody    

The present invention is in one aspect thereof, and provides a humanized anti-CD3 antibody or an antigen-binding fragment thereof.

As for the humanized antibody of the present invention, antibodies that incorporate only complementarity determining regions (CDRs) into human-derived antibodies (Nature (1986) 321, 522-325) can be cited; transplantation by CDR In addition to the sequence of the CDR, a part of the amino acid residues in the framework is also transplanted to the antibody of the human antibody (International Patent Publication No. WO1990 / 007861).

The heavy chain variable region contained in a suitable humanized anti-CD3 antibody or an antigen-binding fragment of the antibody of the present invention holds: a CDRH1 (made of an amino acid sequence shown in SEQ ID NO: 26 (FIG. 24)) GVTFNYYG), and CDRH2 (ITX _aa X _aa GGRI) formed from the amino acid sequence shown in SEQ ID NO: 98 (Figure 112) (where the first X _aa and the second X _aa are each natural Amino acid residues. Hereinafter, the first X _{aa of} CDRH2 is also referred to as X1, and the second X _{aa is also} referred to as X ₁ and X _2. ), And shown by sequence identification number 28 (FIG. 26). CDRH3 (TLDGRDGWVAY).

In addition, the light chain variable region contained in a suitable humanized anti-CD3 antibody or an antigen-binding fragment of the antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 29 (FIG. 27). CDRL1 (TGNIGSNY), and CDRL2 (RX _aa D) formed by the amino acid sequence shown in SEQ ID NO: 99 (Figure 113) (where X _aa is any natural amino acid residue. In the following, we will also refer to X _{aa of} CDRL2 is described as X _3. ), And CDRL3 (QSYSSGFI), which is an amino acid sequence shown in SEQ ID NO: 31 (FIG. 29).

In the above-mentioned CDRH2 (ITX ₁ X ₂ GGRI), preferably X ₁ is selected from the group consisting of (A, E, G, H, I, L, T, V, R, S), and X ₂ Is S; or X ₁ is N, and X ₂ is selected from the group consisting of (E, R, F, Y, L, V, I, K, T), and in the above-mentioned CDRL2 (RX ₃ D) Preferably, X ₃ is selected from the group consisting of (Q, A, G, S, N, D).

In the above-mentioned CDRH2 (ITX ₁ X ₂ GGRI), it is more preferable that X ₁ is selected from the group consisting of (R, S), and X ₂ is S. In the above-mentioned CDRL2 (RX ₃ D), it is more preferably X ₃ is selected from the group consisting of (Q, A, G, S, N, D).

Examples of the heavy chain variable region contained in such a suitable humanized anti-CD3 antibody or antigen-binding fragment of the antibody of the present invention include the amines shown in sequence identification number 100 (FIG. 114). Heavy chain variable regions of amino acid residues.

Examples of the light chain variable region included in a suitable humanized anti-CD3 antibody or an antigen-binding fragment of the antibody of the present invention include sequence identification number 101 (FIG. 115), sequence recognition The light chain variable region of the amino acid residue shown by No. 102 (FIG. 116) and SEQ ID NO: 103 (FIG. 117).

Specific examples of the heavy chain variable region included in a suitable humanized anti-CD3 antibody or an antigen-binding fragment of the antibody of the present invention include an amine represented by SEQ ID NO: 26 (FIG. 24). CDRH1 (GVTFNYYG) based on amino acid sequence, CDRH2 (ITNSGGRI) based on amino acid sequence shown in sequence identification number 27 (Figure 25), and amino group shown in sequence identification number 28 (Figure 26) Heavy chain variable region of CDRH3 (TLDGRDGWVAY) composed of an acid sequence.

In addition, specific examples of the light chain variable region included in a suitable humanized anti-CD3 antibody or an antigen-binding fragment of the antibody of the present invention include those represented by sequence identification number 29 (FIG. 27). CDRL1 (TGNIGSNY) with amino acid sequence, CDRL2 (RDD) with amino acid sequence shown in sequence identification number 30 (Figure 28), and amine shown with sequence identification number 31 (Figure 29) Light chain variable region of CDRL3 (QSYSSGFI) based on amino acid sequences.

In the present invention, the position and length of the CDRs are determined by the definition of IMGT (Developmental and Comparative Immunology 27 (2003) 55-77).

Specific examples of the heavy chain variable region of the present invention include an amino acid sequence included in the amino acid residue shown in SEQ ID NO: 16.

Specific examples of the light chain variable region of the present invention include amino acid sequences included in amino acid residues shown in sequence identification numbers 17, 20, and 23.

As suitable examples of the humanized anti-CD3 antibody or the antigen-binding fragment of the antibody of the present invention, the following antibodies or antigen-binding fragments of the antibody can be enumerated: containing 2 to 119 containing the sequence identification number 60 An amino acid residue of the heavy chain variable region, an antibody comprising a light chain variable region of an amino acid residue of SEQ ID NO: 135 to 243, or an antigen-binding fragment of the antibody, including sequence recognition Antibodies to the heavy chain variable regions of amino acid residues of Nos. 64 to 2 to 119, and antibodies to the light chain variable regions of amino acid residues of SEQ ID NO: 64 to 135 to 241, or the antigen-binding properties of the antibodies A fragment, an antibody comprising a heavy chain variable region comprising an amino acid residue of sequence identification number 2 to 119, an antibody comprising a variable region of a light chain comprising an amino acid residue of sequence identification number 135 to 243, or The antibody has an antigen-binding fragment, a heavy chain variable region containing amino acid residues of SEQ ID NO: 68 to 2 to 119, and a light chain containing amino acid residues of SEQ ID NO: 68 to 135 to 243 Antibodies to variable regions or antigen-binding fragments of the antibodies, comprising a sequence identifier 70 A heavy chain variable region of an amino acid residue of 2 to 119, an antibody to the light chain variable region comprising an amino acid residue of sequence number 70 to 135 to 243, or an antigen-binding fragment of the antibody, An antibody or the antibody comprising a heavy chain variable region comprising an amino acid residue of SEQ ID NO: 72 to 119, and a light chain variable region comprising an amino acid residue of SEQ ID NO: 72 to 135 to 243 Antigen-binding fragment, a heavy chain variable region containing amino acid residues containing sequence identification numbers 74 to 119, and a light chain containing amino acid residues containing sequence identification numbers 74 to 243 are variable Region of the antibody or an antigen-binding fragment of the antibody, a heavy chain variable region comprising an amino acid residue comprising sequence identification number 76 2 to 119, and an amino acid residue comprising an amino acid residue sequence numbering 135 to 243 Antibody or an antigen-binding fragment of the antibody, a heavy chain variable region comprising an amino acid residue comprising sequence identification number 78 2 to 119, and an antibody comprising an antigen-binding fragment thereof and sequence number 135 to An antibody to the light chain variable region of an amino acid residue of 243, or an antigen-binding fragment of the antibody, which contains the sequence identifier 8 Heavy chain variable region of amino acid residues from 2 to 119 of 0, and an antibody or an antigen-binding fragment of the light chain variable region comprising amino acid residues of sequence numbers 80 to 135 to 243 An antibody comprising a heavy chain variable region comprising amino acid residues of sequence identification number 82 2 to 119, an antibody comprising a light chain variable region comprising amino acid residues of sequence identification number 82 135 to 243, or the An antibody-binding fragment of the antibody, or a heavy chain variable region containing an amino acid residue of SEQ ID NO: 84 to 2 to 119, and a light chain containing an amino acid residue of SEQ ID NO: 84 to 135 to 243 An antibody to the variable region or an antigen-binding fragment of the antibody.

In addition, specific examples included in the humanized anti-CD3 antibody or the antigen-binding fragment of the antibody of the present invention include a heavy chain variable region containing an amino acid sequence shown in SEQ ID NO: 16, A linker, an antibody comprising a light chain variable region of an amino acid sequence shown in any one of sequence identification numbers 17, 20, and 23, or an antigen-binding fragment of the antibody.

The variable region contained in the antibody or the antigen-binding fragment of the antibody of the present invention may be combined in the order of a heavy chain variable region and a light chain variable region from the amine terminal side of the antibody, or may be a light chain variable region The sequential binding of the variable regions of the heavy chain. Further, the amino group terminal of the variable region may have a glycine residue, and a linker, a FLAG, and a His tag may be bonded to the carboxy terminal of the variable region.

Examples of suitable specific examples of the humanized anti-CD3 antibody or the antigen-binding fragment of the antibody of the present invention include an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 19 or An antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 22 or an antigen-binding fragment of the antibody, an amine group comprising 2 to 241 of sequence identification number 25 An acid residue antibody or an antigen-binding fragment of the antibody.

As a more suitable specific example contained in the humanized anti-CD3 antibody or the antigen-binding fragment of the antibody of the present invention, an amine group at positions 1 to 243 including the sequence identification number 60 (FIG. 68) may be mentioned. An acid residue antibody (colony strain ID: C3E-7078) or an antigen-binding fragment of the antibody, and an antibody containing an amino acid residue at positions 1 to 241 of sequence identification number 64 (Figure 72) (selection Strain ID: C3E-7085) or an antigen-binding fragment of the antibody, an antibody containing an amino acid residue at positions 1 to 243 of sequence identification number 66 (Figure 74), or (cloned strain ID: C3E-7086) or An antigen-binding fragment of the antibody, an antibody (cloned strain ID: C3E-7087) containing an amino acid residue at positions 1 to 243 of sequence identification number 68 (Figure 76), or an antigen-binding fragment of the antibody, An antibody (cloned strain ID: C3E-7088) containing an amino acid residue at positions 1 to 243 of sequence identification number 70 (FIG. 78) or an antigen-binding fragment of the antibody, including sequence identification number 72 (FIG. 80) ) Antibody (seed ID: C3E-7089) with amino acid residues at positions 1 to 243, or an antigen-binding fragment of the antibody, which contains sequence identification number 74 (FIG. 82) at positions 1 to 243 Amino acid residue Antibody (selected strain ID: C3E-7090) or an antigen-binding fragment of the antibody, and an antibody containing an amino acid residue at positions 1 to 243 of sequence identification number 76 (Figure 84) (selected strain ID) : C3E-7091) or an antigen-binding fragment of the antibody, an antibody (colony strain ID: C3E-7092) containing an amino acid residue at positions 1 to 243 of sequence identification number 78 (Figure 86), or the antibody An antigen-binding fragment, an antibody (cloned strain ID: C3E-7093) containing an amino acid residue at positions 1 to 243 of sequence identification number 80 (Figure 88), or an antigen-binding fragment of the antibody, including the sequence An antibody (selected strain ID: C3E-7094) with an amino acid residue at positions 1 to 243 of the identification number 82 (Fig. 90) or an antigen-binding fragment of the antibody, comprising the sequence identification number 84 (Fig. 92) An antibody having an amino acid residue at positions 1 to 243 (cloned strain ID: C3E-7095) or an antigen-binding fragment of the antibody.

The humanized anti-CD3 antibody or the antigen-binding fragment of the antibody of the present invention binds from the amino acid terminal side in the order of the heavy chain variable region, the first linker, and the light chain variable region, and then Suitable specific examples in which a second linker, a FLAG tag, and a His tag are bound to the carboxy terminus of the light chain variable region include the antigen described in the above (16) or an antigen-binding fragment of the antibody, including: Amino acid sequences containing amino acid residues from 2 to 269 of sequence identification number 22, Amino acid sequences containing amino acid residues from 2 to 267 of sequence identification number 25, 2 to The amino acid sequence of the amino acid residue of 269, the amino acid sequence including the amino acid residues of 2 to 267 of the sequence identification number 64, and the amino acid residue including the amino acid residues of 2 to 269 of the sequence identification number 66 Amino acid sequence, amino acid sequence containing amino acid residues 2 to 269 of sequence identification number 68, amino acid sequence containing amino acid residues of sequence identification number 2 to 269, including sequence identification Amino acid residues of amino acid residues 2 to 269 of number 72, amine groups containing sequence identification numbers 2 to 269 Amino acid sequences of residues, amino acid sequences including amino acid residues of sequence identification number 2 to 269, amino acid sequences including amino acid residues of sequence identification number 2 to 269, An amino acid sequence comprising an amino acid residue of 2 to 269 of sequence identification number 80, an amino acid sequence comprising an amino acid residue of 2 to 269 of sequence identification number 82, or 2 including sequence identification number 84-2 Amino acid sequence to 269 amino acid residues.

For the antibodies and the like of the present invention, the amino acid sequence of the variable region of the heavy chain and / or the amino acid sequence of the variable region of the light chain may be combined with the anti-CD3 antibody of the present invention or the antigen of the antibody 70%, 71%, 72%, 73%, 74%, 75%, 76% of the amino acid sequence of the heavy chain variable region and / or the amino acid sequence of the light chain variable region contained in the sexual fragment , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or 99% or more, and may be a molecule including an antibody that binds to human CD3 and cynomolgus CD3.

The antibody and the like of the present invention can be optimized by introducing mutations into the aforementioned humanized anti-CD3 antibody or the antigen-binding fragment of the antibody, thereby optimizing the ability to bind to CD3. Specific methods for introducing mutations include random mutation induction methods using an error-prone PCR method, site-specific amino acid mutation introduction using the NNK library, and site-specific use of structural information. Mutation introduction and their combinations.

For the antibody and the like of the present invention, in order to reduce the effector activity of the antibody, ADCC and CDC activity may be reduced in place of the constant region.

In order to avoid cytotoxicity to normal human CD3 expressing cells, the effector activity of the antibody is expected to be low. It is known that effector activity varies depending on the subtype of the antibody. It can be seen that IgG4 is low in ADCC and CDC activity. Although IgG2 has CDC activity, ADCC activity is low. With this feature, it is possible to produce antibodies that reduce ADCC and CDC activities by replacing the constant regions of IgG1 with the constant regions of IgG2 and 4. In addition, by referencing IgG2 and 4 and replacing a part of the sequence of the constant region of IgG1, it is possible to produce an IgG1 antibody that reduces ADCC and CDC activities. As an example, according to Marjan Hezareh et.al. Journal of Virology, 75 (24): 12161-12168 (2001), the leucine residues at positions 234 and 235 of IgG1 (numbers are according to Kabat et al. When the EU index) was replaced with an alanine residue, ADCC and CDC activities were reduced.

    (3-8) An antibody that binds to the same site and also binds to cynomolgus CD3    

"Antibodies that bind to the same site and also bind to cynomolgus monkey CD3" provided with the antibodies and the like of the present invention are also included in the antibodies and the like of the present invention. An "antibody bound to the same site" of an antibody means another antibody bound to a site on an antigen molecule recognized by the antibody. If a part of the peptide or part of the three-dimensional structure of the antigen molecule bound by the first antibody is bound by the second antibody, it can be determined that the first antibody and the second antibody are bound at the same site.

In addition, by confirming that the second antibody competes with the first antibody for binding to the antigen, that is, the second antibody hinders the binding of the first antibody to the antigen, even if the peptide sequence or three-dimensional structure of the specific binding site is not determined, the first antibody can be determined. One antibody binds to the same site as the second antibody.

Furthermore, when the first antibody and the second antibody are bound to the same site, and the first antibody has an effect characteristic of one aspect of the antibody of the present invention having cytotoxic activity, the second antibody may have the same activity. Department is extremely high.

Therefore, if a second anti-CD3 antibody binds to the binding site of the first anti-CD3 antibody and the second anti-CD3 antibody binds to cynomolgus monkey CD3, it can be determined that the first antibody and the second antibody bind to the same site on the CD3 protein. . An antibody that binds to the same site on human CD3 that the anti-CD3 antibody or antigen-binding fragment of the antibody binds to and binds to cynomolgus monkey CD3 or an antigen-binding fragment of the antibody is also included in the antibody of the invention. Wait.

If it is confirmed that the second anti-CD3 antibody competes with the first anti-CD3 antibody for binding to CD3 protein, and the second anti-CD3 antibody binds to cynomolgus monkey CD3, it can be determined that the first antibody and the second antibody bind to CD3. Antibodies to the same site on a protein. An antibody that competes with the anti-CD3 antibody of the present invention or an antigen-binding fragment of the antibody on human CD3 and binds to cynomolgus CD3 or an antigen-binding fragment of the antibody is also included in the antibody of the present invention and the like.

The binding site of the antibody can be determined by a method known to those skilled in the art such as an immunoassay. For example, an amino acid sequence of an antigen is prepared as a series of peptides that are appropriately reduced from the carboxyl terminal or the amino terminal. The reactivity of the antibody with these is studied. After roughly identifying the recognition site, a short peptide is further synthesized to study the antibody. Reactivity to these peptides allows the site of binding to be determined. Antigen fragment peptides can be prepared using techniques such as genetic recombination and peptide synthesis.

The antibodies and the like of the present invention recognize and combine to form a three-dimensional structure in CD3, that is, an Ig-like domain. The binding site (antigenic position) of the antibody is determined by X-ray structure analysis by determining the amino acid residue on the antigen adjacent to the antibody.

    (3-9) Modified body of anti-CD3 antibody or antigen-binding fragment thereof    

The present invention provides a modified form of an antibody or an antigen-binding fragment thereof. The modification system of the antibody or the antigen-binding fragment thereof of the present invention means a chemical or biological modification made to the antibody or the antigen-binding fragment thereof of the present invention. The chemical modification includes a bond to a chemical moiety of an amino acid skeleton, a chemical modification of an N-bond or an O-bond carbohydrate chain, and the like. Biological modifications include post-translational modifications (e.g., addition of N- or O-bonded sugar chains, processing of amine-terminal or carboxy-terminal regions, deamination, aspartic acid Isomerization, oxidation of methionine, etc. are manifested by using prokaryotic host cells, and methionine residues are added to the amine end. In addition, in order to make the antibody or antigen of the present invention detectable or isolated, it can be labeled, for example, an enzyme label, a fluorescent label, and an affinity label are also included in the meaning of such a modifier. Such a modified product of the antibody or antigen-binding fragment thereof of the present invention is used to improve stability and blood retention of the antibody or antigen-binding fragment of the original antibody of the present invention, decrease antigenicity, and the antibody or antigen. Checkout or single departure, etc.

Examples of the chemical moiety included in the chemical modification include water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, polyglucose, and polyvinyl alcohol.

Examples of biological modifiers include those that are modified by enzyme treatment, cell treatment, etc., fusions of other peptides such as genetic recombination tags, and sugar chain-modified enzymes that exhibit endogenous or exogenous properties. Cells are used as hosts to modulate.

This modification can be applied to any position in the antibody or its antigen-binding fragment, or to a desired position, and the same or two or more different modifications can be applied to one or two or more positions.

In the present invention, the term "modified body of an antibody-binding fragment of an antibody" also includes "modified body antibody fragment".

In addition, for example, antibodies produced in mammalian cultured cells are known to have a deletion of lysine residues at the carboxy terminus of the heavy chain (Journal of Chromatography A, 705: 129-134 (1995)), and the same heavy chain is known Glycine at the carboxyl terminus and 2 amino acid residues of the lysine are deleted, and the proline residue at the new carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). Furthermore, it is known that in the production of antibodies, the glutamic acid residue or glutamic acid residue of the amine terminal end of the heavy or light chain of the antibody is modified by pyroglutamylation. The anti-system of the present invention may have This modification (International Patent Publication No. WO2013 / 147153).

However, the deletion and modification of these heavy chain sequences have little effect on the antigen-binding ability of the antibody and the effector antigen-binding ability (activation of complement, antibody-dependent cytotoxicity, etc.). Therefore, the present invention also includes the modified antibody and the antigen-binding fragment of the antibody, the deletion body having 1 or 2 amino acid deletions at the carboxy terminus of the heavy chain, and the deletion body (eg, Carboxylated proline residues at the carboxyl-terminated heavy chain), antibodies that have amine terminal residues at the heavy or light chain that are pyroglutaminated, etc. (they are collectively referred to as `` deletions '') . However, as long as all or part of the antigen-binding energy is retained, the carboxyl-terminal deletions of the heavy and light chains of the antibody of the present invention are not limited to the above-mentioned types. When the antibody of the present invention includes two or more chains (for example, a heavy chain), the two or more chains (for example, a heavy chain) may be any heavy chain selected from the group consisting of a full length and the aforementioned deletion body, It may be a combination of two or more. The amount ratio or molecular number ratio of each missing body may be affected by the type and culture conditions of the mammalian culture cells that produce the antibody of the present invention, but examples of the missing body of the antibody of the present invention include two strands. At both ends of the heavy chain, one amino acid residue at the carboxyl terminal may be deleted. All of these are also included in the meaning of the antibody variants, the antigen-binding fragments of the antibodies, or their modifications.

In addition, by regulating the sugar chain modification (glycosylation, deglucosylation, etc.) to which the antibody of the present invention is bound, the antibody-dependent cytotoxic activity can be enhanced. Regarding the technology for regulating sugar chains of antibodies, although international patent publications WO99 / 54342, WO00 / 61739, WO02 / 31140 and the like are known, they are not limited thereto. The antibody of the present invention and the antigen-binding fragment of the antibody also include the sugar chain-modified antibody and the antigen-binding fragment of the antibody.

In the present invention, the scope of the "antibody or antigen-binding fragment thereof" also includes "deletions", "modifications" of "antibody or antigen-binding fragment thereof", and mixtures thereof. In addition, (3-5) and (3-6) describe the range of the "antibody or antigen-binding fragment thereof" included in the antigen-binding molecule, multispecific molecule, and bispecific molecule of the present invention. It also includes "deletions", "modifiers" of "antibodies or antigen-binding fragments thereof", and mixtures thereof.

    4. Manufacturing of antibodies         (4-1) Method using fusion tumor    

As an aspect of the anti-CD3 antibody of the present invention, for example, the method of Kohler and Milstein (Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennet, R .ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)). The spleen of an animal immunized with CD3 protein isolates anti-CD3 antibody-producing cells by spleen the cells from myeloma cells. Fusion, and a fusion tumor is established, and a monoclonal antibody is obtained from a culture of the fusion tumor.

    (4-1-1) Preparation of antigen    

The antigen used to prepare the anti-CD3 antibody can be obtained by a method such as a natural or recombinant CD3 protein (human CD3εγ single chain antigen) preparation method or the like. Examples of the antigens that can be prepared in this manner include CD3 protein or CD3 protein fragments, arbitrary amino acid sequences, and derivatives of additional support (hereinafter, collectively referred to as "CD3").

Natural CD3 can be purified and isolated from, for example, a human tissue-derived cell or a culture of the cell. The recombinant human CD3εγ single-chain antigen system is prepared by introducing a gene containing a base sequence encoding an amino acid sequence possessed by the human CD3εγ single-chain antigen into a host cell, and recovering the antigen from a culture of the cell for modulation. In addition, CD3 obtained by synthesizing a gene contained in the base sequence encoding the amino acid sequence of the CD3 antigen with a cell-free protein by an in vitro translation system is also included in the "CD3 antigen" of the present invention.

    (4-1-2) Production of anti-CD3 monoclonal antibodies    

The production of monoclonal antibodies is usually performed by the following steps.

(a) a step of preparing an antigen, (b) a step of preparing antibody-producing cells, (c) a step of preparing myeloma cells (hereinafter, referred to as "myeloma"), and (d) fusion of antibody-producing cells with myeloma Step (e) a step of selecting a fusion tumor population producing an antibody of interest, and (f) a step of obtaining a single cell line (colony).

If necessary, further (g) a step of culturing the fusion tumor, a step of breeding the animal to which the fusion tumor is transplanted, etc., (h) a step of determining the biological activity of the monoclonal antibody, and a step of judging, etc.

Hereinafter, the method for producing a single antibody is described in detail according to the above steps. However, the method for producing the antibody is not limited to this. For example, antibody-producing cells and myeloma other than spleen cells may be used.

    (a) Steps of preparing antigen    

The CD3 protein of the present invention can be prepared by purifying and isolating animal tissue (including body fluids), cells derived from the tissue, or from the cell culture, genetic recombination, cell-free protein synthesis, chemical synthesis, and the like.

    (b) Steps of modulating antibody-producing cells    

The antigen obtained in step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or potash alum to immunize experimental animals as an immunogen. Experimental animals can be used without any problems in the animals used in the well-known fusion tumor preparation method. Specifically, for example, a mouse, a rat, a sheep, a sheep, a cow, a horse, or the like can be used. However, from the viewpoint of ease of obtaining myeloma cells fused with the extracted antibody-producing cells, it is preferable to use mice or rats as the animals to be immunized.

The strains of mice and rats actually used are not particularly limited. In the case of mice, for example, A, AKR, BALB / c, BALB / cAnNCrj, BDP, BA, CE, C3H, 57BL, C57BL can be used. , C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, 129, etc. In the case of rats, for example, Wistar, Low, Lewis, Sprague-Dawley can be used , ACI, BN, Fischer, etc.

These mouse and rat strains can be obtained, for example, from experimental animal breeding vendors such as Claire Japan, Charles River Japan, and the like.

Among them, considering the suitability for fusion with myeloma cells to be described later, it is particularly preferable to use BALB / c strains as mice to be immunized in mice, and to use Wistar and Low strains as immunized animals in rats.

In addition, considering the identity of the antigen to humans and mice, it is preferable to use mice with autoimmune diseases in order to reduce the biological mechanism of autoantibody removal.

Moreover, the age of the mice or rats at the time of immunization is preferably 5 to 12 weeks, more preferably 6 to 8 weeks.

For immunizing animals with CD3 protein, for example, Weir, DM, Handbook of Experimental Immunology Vol.I.II.III., Blackwell Scientific Publications, Oxford (1987), Kabat, EA and Mayer, MM, Experimental Immunochemistry, Charles C Thomas Publisher Spigfield, Illinois (1964), etc.

Examples of the method for measuring the antibody value include, but are not limited to, such immunoassays as RIA method and ELISA method.

Spleen cells or lymphocyte-derived antibody-producing cells isolated from immunized animals can be prepared according to, for example, Kohler et al., Nature (1975) 256, 495; Kohler et al., Eur. J. Immnol. (1977 ) 6,511 ,; Milstein et al., Nature (1977), 266, 550 ,; Walsh, Nature (1977) 266, 495, etc.) for modulation.

In the case of spleen cells, a general method of separating antibody-producing cells by finely cutting the spleen, filtering the cells through a stainless steel mesh, and floating the cells on Eagle minimum essential medium (MEM) can be used.

    (c) Steps for modulation of myeloma    

Myeloma cells used for cell fusion are not particularly limited, and can be appropriately selected and used from known cell lines. Considering the convenience of selecting fusion tumors from fused cells, it is preferred to use the established procedures for selection (hypoxanthine). -Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) -deficient strain, that is, mouse-derived X63-Ag8 (X63), NS1-ANS / 1 (NS1), P3X63-Ag8.U1 (P3U1) , X63-Ag8.653 (X63.653), SP2 / 0-Ag14 (SP2 / 0), MPC11-45.6TG1.7 (45.6TG), FO, S149 / 5XXO, BU.1, etc. 210.RSY3.Ag.1.2.3 (Y3), etc., human-derived U266AR (SKO-007), GM1500‧GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), 8226AR / NIP4 -1 (NP41) and so on. These HGPRT-deficient strains can be obtained from, for example, the American Type Culture Collection (ATCC).

These cell lines are prepared in a suitable medium, such as 8-azaguanine medium (with glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine serum (hereinafter referred to as "" FCS ") medium with 8-azaguanine], Iscove's modified DEME (Iscove's Modified Dulbecco's Medium; hereinafter referred to as" IMDM "), or Dulbecco's Modified Eagle Medium; hereinafter referred to as" DMEM ") for subculture, but 3 to 4 days before cell fusion, subculture in normal medium [for example, ASF104 medium containing 10% FCS (manufactured by Ajinomoto Co., Ltd.)], and make sure on the day of fusion There are 2 × 10 ⁷ or more cells.

    (d) the step of fusing antibody-producing cells with myeloma cells    

The fusion of antibody-producing cells and myeloma cells is according to a known method (Weir, DM, Handbook of Experimental Immunology Vol.I.II.III., Blackwell Scientific Publications, Oxford (1987), Kabat, EA and Mayer, MM, Experimental Immunochemistry , Charles C Thomas Publisher Spigfield, Illinois (1964), etc.) can be implemented under conditions where the survival rate of the cells is not extremely reduced. For example, a chemical method of mixing antibody-producing cells with myeloma cells in a high-concentration polymer solution such as polyethylene glycol, a physical method using electrical stimulation, and the like can be used.

    (e) a step of selecting a fusion tumor population producing the antibody of interest    

The selection method of fusion tumors obtained by cell fusion is not particularly limited, but HAT (hypoxanthine aminopterin thymidine) selection method is generally used (Kohler et al., Nature (1975) 256, 495; Milstein et al., Nature (1977) 266, 550). This method is effective when a fusion tumor is obtained by using myeloma cells of an HGPRT-deficient strain in which aminopterin cannot survive. That is, by culturing unfused cells and fused tumors in a HAT medium, fusion tumors that are only resistant to aminopterin can be selectively left and can proliferate.

    (f) Steps to obtain a single cell line (colony)    

As for the colony selection method of the fusion tumor, for example, a known method such as a methyl cellulose method, a soft agarose method, and a limiting dilution method (for example, Barbara, BM and Stanley, MS: Selected Methods in Cellular Immunology, WH) can be used. Freeman and Company, San Francisco (1980)), preferably the limiting dilution method.

    (g) Culture steps of fusion tumors, breeding steps of animals transplanted with fusion tumors    

By culturing the selected fusion tumor, a single antibody can be produced, and preferably, the desired fusion tumor is cloned and supplied to the production of the antibody.

The single-antibody system that produces the fusion tumor can be recovered from the culture of the fusion tumor. Furthermore, a recombinant antibody can be recovered from a culture of cells into which the single antibody gene has been introduced. Furthermore, the fusion tumor can be injected into the abdominal cavity of mice of the same strain (for example, the above-mentioned BALB / cAnNCrj) or Nu / Nu mice, and the fusion tumor can be recovered from the ascites by proliferating the fusion tumor.

    (h) Measurement steps and determination steps for the biological activity of individual antibodies    

Various biological tests can be selected and applied according to the purpose.

    (4-2) Cellular Immunoassay    

By using cells expressing natural CD3, cells expressing recombinant CD3 or fragments thereof as immunogens, anti-CD3 antibodies can be prepared by the aforementioned fusion tumor method.

Examples of cells expressing natural CD3 include human thymocytes and T lymphocytes. Their CD3 expressing cell lines use 1 × 10 ⁵ to 1 × 10 ⁹ cells at a time, preferably 1 × 10 ⁶ to 1 × 10 ⁸ cells, more preferably 0.5 to 2 × 10 ⁷ cells, and even more preferably 1 × 10 ⁷ cells, but the number of cells supplied to the immune system can be changed according to the expression of CD3. The immune source is generally administered into the abdominal cavity, or it can be administered into the skin or the like. As for the production method of the fusion tumor, the method described in (4-1-2) can be applied.

    (4-3) DNA Immunoassay    

The anti-CD3 antibody of the present invention can also use DNA immunoassay. An antigen-expressing plastid gene is introduced into an individual animal such as a mouse or a rat, and the antigen is expressed in the individual to induce immunity to the antigen. For gene introduction, there are methods for directly injecting plastids into muscles, methods for injecting liposomes or polyethyleneimine into test substances, intravenous methods, methods using viral vectors, and using gene guns to make adherents The method of injecting gold particles into the body, the hydrodynamic method of rapidly injecting a large amount of plastid solution as an intravenous injection, and the like.

As an example of the rat anti-human CD3 antibody thus established, C3-147 can be cited. The amino acid sequence of the light chain variable region of C3-147 is shown in SEQ ID NO: 9 in the Sequence Listing (Figure 17). The amino acid sequence of the heavy chain variable region of C3-147 is shown in SEQ ID NO: 7 in the Sequence Listing (Figure 15).

    (4-4) Genetic recombination    

The antibody of the present invention comprises a nucleotide (heavy chain nucleotide) contained in a nucleotide sequence encoding a heavy chain amino acid sequence and a core contained in a nucleotide sequence encoding a light chain amino acid sequence thereof. Nucleotide (light chain nucleotide), or a vector with the heavy chain nucleotide inserted and a vector with the light chain nucleotide inserted, are introduced into a host cell, and after the cell is cultured, the antibody can be recovered from the culture. To modulate. Heavy chain and light chain nucleotides can be inserted into one vector.

As the host cell, a prokaryotic cell or a eukaryotic cell can be used. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used.

As the animal cell, for example, mammalian-derived cells, that is, human fetal-derived kidney cells HEK293F cells (Subedi GP et al., J Vis Exp. (2015) 106) derived from monkey kidney. COS Cells (Gluzman, Y. Cell (1981) 23, 175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658), Chinese hamster ovary cells (CHO cells, ATCC CCL-61) 2. Its dihydrofolate reductase-deficient strain (CHOdhfr-: Urlaub, G. and Chasin, LAPNAS (1980) 77, 4126-4220), cells derived from birds such as chickens, cells derived from insects, and the like.

In addition, cells that change to increase the biological activity of the antibody by changing the structure of the sugar chain can also be used as a host. For example, among N-glycoside-binding complex sugar chains bound to the Fc region of an antibody, the ratio of sugar chains that are not bound to fucose by N-acetylglucosamine used at the reducing end of the sugar chain is changed to 20%. The above CHO cells can modulate antibodies with increased ADCC activity and CDC activity (International Patent Publication No. WO02 / 31140).

Examples of eukaryotic microorganisms include yeast and the like. Examples of the prokaryotic cells include coliform, subtilis, and the like.

The information peptides used to secrete the antibodies (single-body antibodies, rat antibodies, mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies, etc. derived from various animals) of the present invention are not limited to those related to The secretion information of the antibody isotype, isotype and isotype antibody, and the secretion information of the antibody itself, if it is the secretion information of other type or subtype antibody, or the protein derived from other eukaryote species or prokaryotes The secretion information can be selected and used.

Antibodies and the like that are secreted by including a message peptide are also included in the antibodies and the like of the present invention and molecules of the present invention.

    (4-5) Design and preparation of humanized antibodies    

Examples of humanized antibodies include antibodies in which only CDRs of non-human animal antibodies are incorporated into human-derived antibodies (see Nature (1986) 321, p. 522-525), and sequences added to the CDRs by the CDR grafting method. Some of the amino acid residues of the framework are also transplanted to human antibody antibodies (see WO90 / 07861, US6972323), and one or more amino acid residues of non-human animal antibodies in any of them Human-type amino acid-substituted antibodies and the like are not limited to them.

    (4-6) Preparation of human antibodies    

Examples of the antibody of the present invention include human antibodies. The human anti-CD3 antibody system means an anti-CD3 antibody derived from the amino acid sequence of a human-derived antibody. The human anti-CD3 antibody system is a method for producing a mouse by using a human antibody comprising a human genomic DNA fragment containing genes having a heavy chain and a light chain of a human antibody (see Tomizika, K. et al., Nature Genetics (1997) 16 , 133-143 ,; Kuroiwa, Y.et.al., Nuc. Acids Res. (1998) 26, 3447-3448; Yoshida, H.et.al., Animal Cell Technology: Basic and Applied Aspects vol.10, 69-73 (Kitagawa, Y., Matuda, T. and Iijima, S.eds.), Kluwer Academic Publishers, 1999 .; Tomizika, K.et.al., Proc. Natl. Acad. Sci. USA (2000) 97, 722-727, etc.).

Specifically, such human antibody-producing animals can be introduced into yeast artificial chromosome (YAC) vectors by disrupting the loci of the immunoglobulin heavy and light chains inherent in non-human mammals. Human immunoglobulin heavy and light chain loci. In addition, by genetic recombination technology, eukaryotic cells are transformed with cDNAs encoding the heavy and light chains of such human antibodies, preferably vectors containing the cDNA, and genetically recombinant human individual strains are produced by culture. The transformed cells of the antibody can also be obtained from the culture supernatant.

Among these, eukaryotic cells can be used as the host, and mammalian cells such as HEK293F cells and CHO cells are preferably used.

In addition, a method for obtaining a phage-displayed human antibody screened from a free human antibody library is also known. For example, a phage display method in which a variable region of a human antibody is expressed as a scFv on the surface of a phage and a phage selected for binding to an antigen can be used. By analyzing the genes of the phage selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of the scFv that binds to the antigen is clear, a expression vector with the sequence can be prepared and introduced into an appropriate host for expression, and human antibodies (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, 433-455).

    (4-7) Preparation method of antigen-binding fragment of antibody    

Methods for creating scFvs are well known in the art (for example, refer to US Patent No. 4,946,778, US Patent No. 5,260,203, US Patent No. 5,091,513, US Patent No. 5,455,030, etc.). Here, in scFv, the variable region of the heavy chain and the variable region of the light chain are linked by a linker, preferably by a polypeptide linker, without making a conjugate (Huston, JSet al., PNAS (1988) , 85, 5879-5883). The heavy chain variable region and light chain variable region in scFv may be derived from the same antibody or may be derived from separate antibodies.

As the polypeptide linker to which the variable region is linked, for example, any single-chain peptide composed of 5 to 30 residues can be used.

The scFv-encoding DNA is the entire or desired amino acid sequence encoding the DNA encoding the heavy or heavy chain variable region of the aforementioned antibody, and the DNA encoding the light or light chain variable region, and their sequences. The DNA portion is used as a template, and designated primers are used to amplify both ends by PCR method. Then, the DNA encoding the polypeptide linker portion is combined, and the primers connected to the heavy and light chains at each end are combined. And get. Alternatively, it can also be obtained by completely synthesizing DNA encoding the entire region of scFv.

Using scFv-encoding DNA, a expression vector containing the DNA and a host cell transformed with the expression vector can be prepared according to a usual method, and the scFv can be recovered from the culture by culturing the host cell according to a usual method. .

Antigen-binding fragments of other antibodies can also be obtained by introducing a gene encoding the antigen-binding fragment into a cell according to the aforementioned method, and recovering the antigen-binding fragment from a culture of the cell.

The antibody and the like of the present invention can be quantified to increase the affinity for the antigen. A multi-quantitative antibody may be a type of antibody or a plurality of antibodies that recognize multiple epitopes of the same antigen. Methods for quantifying antibodies include the binding of IgG CH3 domain to two scFvs, binding to streptavidin, introduction of helix-turn-helix motif, etc. .

The antibodies and the like of the present invention may be a mixture of a plurality of types of anti-CD3 antibodies having different amino acid sequences, that is, multiple antibodies. For multiple antibodies, for example, a part or all of the CDR group is a mixture of different plural kinds of antibodies. Such multiple strains of antibodies can be used to culture different antibody-producing cells in a mixed culture and recovered from the culture (WO2004 / 061104). Moreover, it is also possible to mix individually prepared antibodies. In addition, an antiserum that is one aspect of a plurality of antibodies can immunize an animal with a desired antigen, and can be prepared by recovering serum from the animal according to a usual method.

As a modified product of an antibody, an antibody that binds to various molecules such as polyethylene glycol (PEG) can also be used.

The antibody or the like of the present invention may be a complex (Immunoconjugate) in which these antibodies and other molecules are linked by a linker. Examples of the antibody-drug complex in which the antibody binds to a radioactive substance or a compound (drug) having a pharmacological action include ADC (Antibody-Drug Conjugate) (Methods MolBiol. (2013) 1045: 1-27).

The antibodies and the like of the present invention can further associate these antibodies with other functional polypeptides. Examples of such an antibody-peptide complex include a complex of the antibody and an albumin-binding polypeptide (Protein Eng Des Sel. (2012) (2): 81-8).

    (4-8) Purification of antibodies and antigen-binding fragments of antibodies    

The obtained antibody and the antigen-binding fragment of the antibody can be uniformly purified in a manner not including the antibody. The isolation and purification of the antibody and the antigen-binding fragment of the antibody may be performed using isolation and purification methods commonly used for proteins.

For example, antibodies can be separated and purified by appropriate selection, combination of chromatography columns, filtration, ultrafiltration, salting-out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric point electrophoresis, etc., but it is not limited to Wait.

For a better isolation and purification method, for example, a expression vector is prepared by attaching a DNA sequence encoding a His tag or a FLAG tag to the carboxy terminus of an antibody variable region, and the vector is used to transform cells and further culture. The cell and the antigen-binding fragment of the antibody are expressed. After the culture is completed, the culture supernatant is extracted and subjected to metal affinity chromatography such as Ni, Co, anti-FLAG tag antibody column, colloid filtration, ion exchange chromatography, etc. Can be purified.

Antibodies and antigen-binding fragments of antibodies expressed by amino acid sequences encoding tags such as His tags or FLAG tags are also included in the antibodies and the like of the present invention and the molecules of the present invention.

    (4-9) Multispecific molecules, bispecific molecules    

The bispecific molecule and multispecific molecular system of the present invention can be exemplified by a method for introducing a plastid into a host cell for temporary expression; after the plastid is introduced into the host cell, a stable and stable cell line is selected and selected by an agent, and A method of constant expression; a method of cell-free synthesis; a method of chemically binding each antibody or antigen-binding fragment by the above method using a synthetic peptide linker.

For bispecific molecules that use the variable region of an antibody, there is a peptide linker that binds a 1-strand chain antibody (scFv) to each other (scFv in tandem) and exchanges the domains of the two antibodies with different specificities instead of Covalently bond to form a dimer (double-chain antibody), and two antibodies with different specificities exchange their domains and single-chain (single-chain double-chain antibody). Non-covalently bonded dimer (TandAb, US Pat. No. 7,129,330) and the like.

The present invention also provides an antibody of the present invention or an antigen-binding fragment of the antibody, or a gene encoding a modified substance such as an antigen, a recombinant vector having the gene inserted therein, a cell into which the gene or vector is introduced, and other antibodies producing the present invention. Cell.

    5. Pharmaceutical composition    

The present invention provides an anti-CD3 antibody, an antigen-binding fragment or a modification thereof, and / or a molecule of the present invention containing them, for example, a pharmaceutical composition containing a multispecific molecule.

In the present invention, the treatment and / or prevention of a disease includes the prevention, aggravation, or inhibition of the onset of the disease, the amelioration, aggravation, or inhibition of one or two or more symptoms exhibited by an individual suffering from the disease. Relief, treatment or prevention of secondary diseases, etc., but not limited to them. In terms of diseases, for example, in the case of the aforementioned molecules, cancer can be cited.

The pharmaceutical composition of the present invention may contain a therapeutically or prophylactically effective amount of an anti-CD3 antibody or an antigen-binding fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or Adjuvant.

A "therapeutically or prophylactically effective amount" means an amount that exerts a therapeutic or preventive effect on a specific disease, form of administration, and route of administration.

The pharmaceutical composition of the present invention may contain pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, solubility, and release properties of the composition or the antibodies contained therein. , Absorptivity, permeability, dosage form, strength, properties, shape and other changes, maintenance, retention substances (hereinafter referred to as "agents for preparations"). The substance for the preparation is not particularly limited as long as it is a pharmacologically acceptable substance. For example, non-toxic or low-toxic substances are preferred properties.

Examples of the substances used in the preparation include, but are not limited to, the following; glycine, alanine, glutamic acid, aspartic acid, histidine, arginine, or lysine Amino acids such as acids, antibacterial agents, ascorbic acid, sodium sulfate or sodium bisulfite, etc. antioxidants, phosphoric acid, citric acid, boric acid buffers, sodium bicarbonate, Tris-Hcl solution, etc. , Fillers such as mannitol or glycine, chelating agents such as ethylenediaminetetraacetic acid (EDTA), caffeine, polyvinyl pyrrolidine, β-cyclodextrin or hydroxypropyl-β-cyclodextrin, etc. Additives, extenders such as glucose, mannose or dextrin, monosaccharides, disaccharides or other carbohydrates such as glucose, mannose or dextrin, colorants, flavoring agents, diluents, emulsifiers or polyethylene Hydrophilic polymers such as pyrrolidine, low-molecular-weight peptides, salt-forming para-ions, benzyl ammonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl parahydroxybenzoate, propyl parahydroxybenzoate, Luo Preservatives such as chlorhexidine, sorbic acid or hydrogen peroxide, sugar Oil, solvent such as propylene glycol or polyethylene glycol, sugar alcohol such as mannitol or sorbitol, suspension agent, PEG, sorbitan ester, polysorbate 20 or polysorbate 80 , Triton, tromethamine, surfactants such as lecithin or cholesterol, stabilization enhancers such as sucrose or sorbitol, sodium chloride, potassium chloride, mannitol or sorbitol Elasticity enhancers such as sugar alcohols, delivery agents, diluents, excipients, and / or pharmaceutical adjuvants.

The amount of these agents for preparations is 0.001 to 1,000 times the weight of the anti-CD3 antibody, its antigen-binding fragment or modification thereof, or the molecule of the present invention, for example, a multispecific molecule, preferably 0.01 to 100 times, more preferably 0.1 to 10 times.

Antibodies comprising the anti-CD3 antibody of the present invention, an antigen-binding fragment or modification thereof, or a molecule of the present invention, for example, an immunoliposome in which a multispecific molecule is contained in a liposome, and an antibody is combined with the liposome The pharmaceutical composition of the modified body (US Patent No. 6,214,388, etc.) is also included in the pharmaceutical composition of the present invention.

The excipient or carrier is usually a liquid or a solid, as long as it is water for injection, physiological saline, artificial cerebrospinal fluid, or other preparations for oral or parenteral administration, and is not particularly limited. limited. Examples of the physiological saline include those who are neutral and those who include serum albumin.

As the buffering agent, a Tris buffer solution having a final pH of 7.0 to 8.5, a acetic acid buffer solution having a pH of 4.0 to 5.5, a citric acid buffer solution having a pH of 5.0 to 8.0, and a pH adjusting agent can be exemplified. 5.0 to 8.0 histidine buffer and the like.

The pharmaceutical composition of the present invention is a solid, liquid, suspension, or the like. Examples include freeze-dried preparations. Excipients such as sucrose can be used to shape the freeze-dried formulation.

The administration route of the pharmaceutical composition of the present invention may be any of enteral administration, local administration, and parenteral administration, and an appropriate administration route may be selected according to the target disease. Specific examples include intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, percutaneous administration, intraosseous administration, intra-articular administration, etc. .

The composition of the pharmaceutical composition can be determined according to the administration method, the CD3 protein binding affinity of the antibody, and the like.

The present invention is an anti-CD3 antibody, an antigen-binding fragment or a modification thereof, or a molecule of the present invention. For example, the amount of a multispecific molecule administered may be based on the type of the individual, the type of the disease, the symptoms, the sex, the age, Elderly disease, the CD3 protein binding affinity of the antibody or its biological activity and other factors are appropriately determined, but it is usually 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg, once every 1 to 180 days, or 1 Administer 2 or 3 times a day.

Examples of the form of the pharmaceutical composition include injections (including freeze-dried preparations, drips), suppositories, nasal absorption preparations, percutaneous absorption preparations, sublingual preparations, capsules, lozenges, ointments, and granules. , Aerosols, pills, powders, suspensions, emulsions, eye drops, implantable preparations, etc.

The present invention is an anti-CD3 antibody, an antigen-binding fragment or a modification thereof, and / or a molecule of the present invention containing them, for example, a multispecific molecule (hereinafter, referred to as an anti-CD3 antibody, etc.) may be combined with other agents. use. An anti-CD3 antibody or the like or a pharmaceutical composition containing the same as an active ingredient may be administered simultaneously or separately with other pharmaceutical ingredients containing an agent other than the anti-CD3 antibody as an active ingredient. For example, after the other agent is administered, a pharmaceutical composition containing an anti-CD3 antibody or the like as an active ingredient may be administered, or after a pharmaceutical composition containing an anti-CD3 antibody or the like as an active ingredient is administered, the other agent may be administered, or the anti-CD3 antibody or the like may be administered. A pharmaceutical composition such as a CD3 antibody as an active ingredient is administered simultaneously with other agents. Either the case where an active ingredient of both anti-CD3 antibodies and other agents are contained in a single pharmaceutical composition, or the case where both of the active ingredients are contained in a plurality of pharmaceutical compositions are referred to in the present invention as " Pharmaceutical composition containing anti-CD3 antibodies and other agents. " In the present invention, the "pharmaceutical composition" has the same meaning as "a pharmaceutical composition in which an anti-CD3 antibody or the like is administered in combination with other agents".

In the present invention, the "combination administration" of an anti-CD3 antibody and other agents means that the anti-CD3 antibody and other agents are incorporated into the body of a subject to be administered for a certain period of time. Anti-CD3 antibodies and other agents may be administered as preparations contained in a single preparation, or they may be formulated separately and administered separately. In the case of individual preparations, the administration period is not particularly limited, and they may be administered simultaneously, or they may be administered at different times or on different days at intervals. When the anti-CD3 antibody and other agents are administered at different times or days, the order of administration is not particularly limited. Generally, the respective preparations are administered in accordance with their respective administration methods, and their administration may be the same number of times or may be different times. In addition, in the case where each is formulated separately, the administration method (administration route) of each preparation may be the same or may be administered by a different administration method (administration route). In addition, it is not necessary that the anti-CD3 antibody and the like are stored in the body at the same time as other agents, as long as it is taken into the body within a certain period (for example, one month, preferably one week, more preferably several days, and even more preferably one day). It can also disappear from the body when the other one is administered.

Examples of the administration form of the "pharmaceutical composition in which an anti-CD3 antibody and other agents are administered in combination" include, for example, 1) administration of a single preparation containing an anti-CD3 antibody and other agents, and 2) administration of an anti-CD3 antibody and other agents. CD3 antibody and other two preparations obtained by separate preparation of other agents are administered simultaneously in the same administration route, 3) anti-CD3 antibody and other preparations obtained by separate preparation of other agents are the same Administration of time difference between administration routes, 4) simultaneous administration of two preparations obtained by separately formulating anti-CD3 antibodies and other agents, and 5) administration of anti-CD3 antibodies and other agents Administration of two preparations obtained by respective preparations, such as administration at different time intervals between administration routes. The administration amount, administration interval, administration form, preparation, etc. of the "pharmaceutical composition in which anti-CD3 antibodies and other agents are administered in combination" are similar to the administration amounts of pharmaceutical compositions containing anti-CD3 antibodies, The administration interval, administration form, preparation, etc. are not limited to them.

In the case where the pharmaceutical composition becomes two different preparations, the pharmaceutical composition may be a set including them.

In the present invention, the "combination" of the anti-CD3 antibody and the like and other agents means the "combination and administration" of the anti-CD3 antibody and the like and other agents.

The combination or pharmaceutical composition of the present invention can further use other medicines.

The present invention also provides a method for treating or preventing a disease related to CD3, the use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease, and the use of the antibody of the present invention for treating or preventing the disease. . A therapeutic or preventive kit comprising the antibody of the present invention is also included in the present invention.

    [Example]    

Hereinafter, the present invention will be described more specifically, but the present invention is not limited to them. In addition, in the following examples, as long as each operation regarding genetic manipulation is not specifically stated, the term "Molecular Cloning" (Sambrook, J., Fritsch, EF, and Maniatis, T., Cold Spring Harbor Laboratory Press published in 1989), or when using commercially available test drugs or kits, use them according to the instructions of the commercially available products.

    (Example 1) Preparation of rat anti-human CD3 antibody         1) -1 Construction of human CD3εδ expression vector    

Using the Gateway Vector Conversion System (Thermo Fisher Scientific), a control vector pcDNA3.1-DEST changed to a destination vector was produced. The cDNA encoding the human CD3ε protein (NCBI Reference Sequence: NP_000724.1) shown in Figure 1 (sequence identification number 1) was purchased from Sino Biological, using the Gateway LR Clonase Enzyme mix (Thermo Fisher Scientific), and was cloned in pcDNA3. 1-DEST vector to construct hCD3ε-pcDNA3.1. The cDNA encoding the human CD3δ protein (NP_000723.1) shown in FIG. 2 (sequence identification number 2) is a PCR method using a human T cell-derived cDNA as a template according to a method known to those skilled in the art. The expression vector hCD3δ-pcDNA3.1 was constructed by breeding with pcDNA3.1 (+) (Thermo Fisher Scientific). For mass modulation of each expression vector, Endofree Plasmid Giga Kit (QIAGEN) was used.

    1) -2 immunity    

For the immune system, female WKY / Izm rats (Japan SLC) were used. First, preliminarily treat the calf of both legs of rats with hyaluronidase (SIGMA-ALDRICH), and then intramuscularly inject hCD3ε-pcDNA3.1 and hCD3δ-pcDNA3.1 prepared in Example 1) -1 at the same site. Performance vector. Next, in vivo electroporation was performed on the same site using ECM830 (BTX) using two needle electrodes. After repeating in vivo electroporation about once every two weeks, rat lymph nodes or spleens were collected and used for fusion tumor production

    1) -3 fusion tumor production    

Lymph node cells or spleen cells were combined with mouse myeloma SP2 / 0-ag14 cells (ATCC, No. CRL-1 581) by electric cell fusion using LF301 Cell Fusion Unit (BEX), and ClonaCell-HY Selection Medium D (StemCell Technologies) was diluted and cultured. A single fused tumor was produced by recovering the emerged fused tumor community. Each recovered fusion tumor strain was cultured using ClonaCell-HY Selection Medium E (StemCell Technologies), and the obtained fusion tumor culture supernatant was used to screen for fusion tumors produced by anti-human CD3 antibodies.

    1) -4 Antibody screening using Cell-ELISA         1) -4-1 Cell-ELISA Modulation of Cells Expressed by Antigen Genes    

HEK293α cells (a stable expression cell line derived from HEK293 expressing integrin αv and integrin β3) were prepared at 7.5 × 10 ⁵ cells / mL in DMEM medium containing 10% FBS. For this, according to the transformation procedure using Lipofectamine 2000 (Thermo Fisher Scientific), introduce hCD3ε-pcDNA3.1 and hCD3δ-pcDNA3.1 or pcDNA3.1-DEST as a control, and aliquot 100 μL each into a 96-well plate ( Corning), and cultured in a DMEM medium containing 10% FBS at 37 ° C and 5% CO ₂ overnight. The obtained introduced cells were used in a cell-ELISA state as they were.

    1) -4-2 Cell-ELISA    

After the expression vector prepared in Example 1) -4-1 was introduced into the culture supernatant of HEK293α cells, the hCD3ε-pcDNA3.1 and hCD3δ-pcDNA3.1 or pcDNA3.1-DEST were introduced into HEK293α cells to add fusion. The tumor culture supernatant was left at 4 ° C for 1 hour. After washing the cells in the wells once with 5% FBS-containing PBS, anti-rat IgG and HRP-Linked Whole Ab Goat diluted 500-fold with 5% FBS-containing PBS was added. (GE Healthcare Bioscience), and allowed to stand at 4 ° C for 1 hour. After the cells in the wells were washed twice with 5% FBS in PBS, OPD color solution (OPD solution (0.05M 3 sodium citrate, 0.1M sodium hydrogen phosphate ‧ 12 water, pH 4) was added at 100 μL / well. 5) In each case, o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) and H ₂ O _{2 were} dissolved to 0.4 mg / mL, 0.6% (v / v)). The color development reaction was carried out while stirring occasionally, and 1 M HCl was added at 100 μL / well to stop the color development reaction, and then the absorbance at 490 nm was measured with a plate reader (ENVISION: PerkinElmer). In order to select fusion tumors that produce antibodies that bind to human CD3 expressed on the cell membrane surface, compared with the control pcDNA3.1-DEST introduced into HEK293α cells, the hCD3ε-pcDNA3.1 and hCD3δ-pcDNA3.1 expression vectors were introduced into HEK293α cells Fusion tumors producing culture supernatants showing higher absorbance were positive as anti-human CD3 antibodies.

    1) -5 antibody screening using human T cell activation    

The activation of T cells by the anti-CD3 antibody produced by the obtained fusion tumor was evaluated using the detection of a CD69 activation marker as an index. Jurkat cells (ATCC, No. TIB-152), which is a human T cell line, were prepared in a RPMI1640 medium containing FBS to a concentration of 5 × 10 ⁶ cells / mL, and 100 μL of each was seeded in a 96-well plate. In Jurkat cells from which the supernatant was removed by centrifugation, Cell-ELISA added to Example 1) -4 at a final concentration of 5 μg / mL was selected as the culture supernatant of fusion tumors producing positive anti-human CD3 antibodies, or rats. An IgG isotype control antibody (R & D Systams) was left at 37 ° C for 30 minutes. Then, cross-linked goat anti-rat IgG FcγFragment specific (JACKSON IMMUNORESEARCH) was added at a final concentration of 10 μg / well, and cultured overnight at 37 ° C and 5% CO ₂ . On the next day, the supernatant was removed, the cells in the wells were washed once with 5% FBS in PBS, and then PE mouse anti-human CD69 antibody (BD Bioscience) was added at 20 μL / well, and allowed to stand at 4 ° C for 30 minutes. minute. The cells in the wells were washed twice with 5% FBS-containing PBS, resuspended in 5% FBS-containing PBS, and detected by a flow cytometer (FC500: Beckman Coulter). Data analysis was performed using Flowjo (Treestar). A histogram of the fluorescence intensity of PE was prepared, and a fusion tumor of a sample in which the histogram of the fluorescence intensity of PE produced relative to the fluorescence intensity histogram of the rat IgG isotype control antibody was shifted to the side of strong fluorescence intensity was selected as human T Cell activation can produce fusion tumors with positive anti-human CD3 antibodies.

    1) -6 Selective binding screening for human or monkey CD3 using flow cytometry         1) Modulation of -6-1 human antigen gene expression cells    

Lenti-X293T cells (TAKARA, Cat # 632180) were seeded in a 225 cm ² flask to become 5.3 × 10 ⁴ cells / cm ² , and cultured in a DMEM medium containing 10% FBS at 37 ° C and 5% CO ₂ one night. On the next day, hCD3ε-pcDNA3.1 and hCD3δ-pcDNA3.1, or pcDNA3.1-DEST as a control, were each introduced into Lenti-X293T cells using Lipofectamine 2000, and further cultured at 37 ° C under 5% CO ₂ late. The following day, the expression vector was introduced into Lenti-X293T cells and treated with TrypLE Express (Thermo Fisher Scientific). The cells were washed with DMEM containing 10% FBS, and then prepared into 5 × 10 ⁶ cells / PBS with 5% FBS. Concentration in mL. The obtained cell suspension was analyzed by flow cytometry.

    1) Analysis of -6-2 binding to human CD3 by flow cytometry    

Furthermore, the specificity of human CD3 binding antibodies produced by fusion tumors that were determined to be positive for human T cell activation in Example 1) -5 was confirmed by flow cytometry. The Lenti-X293T cell suspension prepared in Example 1) -6-1 was inoculated at a rate of 100 μL / well to a 96-well U-bottom microtiter plate, and the supernatant was removed after centrifugation. HCD3ε-pcDNA3.1 and hCD3δ-pcDNA3.1 were introduced into Lenti-X293T cells and pcDNA3.1-DEST were introduced into Lenti-X293T cells, respectively. The fusion tumor culture supernatant was added to the suspension, and the suspension was left at 4 ° C for 1 hour. After washing once with 5% FBS-containing PBS, an anti-rat IgG FITC conjugate (SIGMA) diluted 500-fold with PBS containing 5% FBS was added and suspended, and left to stand at 4 ° C for 30 minutes. After washing twice with 5% FBS-containing PBS, the suspension was resuspended in 5% FBS-containing PBS containing 2 μg / ml 7-aminoactinomycin D (Molecular Probes), and flow-through Cytometer for detection. Data analysis was performed using Flowjo. After 7-aminoradidin D-positive dead cells were excluded by the gate, a histogram of FITC fluorescence intensity of living cells was made. Choose to generate histograms of fluorescence intensities relative to pcDNA3.1-DEST introduced into Lenti-X293T cells as a control, and histograms of hCD3ε-pcDNA3.1 and hCD3δ-pcDNA3.1 into Lenti-X293T cells shifted to strong fluorescence intensity The fusion tumor on the side of the sample produces a fusion tumor as an antibody that binds to human CD3.

    1) Construction of 6-3 monkey CD3εδ expression vector    

CDNA lines encoding monkey CD3ε protein (NCBI reference sequence: NP_001270544.1) and monkey CD3δ protein (NCBI reference sequence: NP_001274617.1) Those with ordinary knowledge in the technical field according to known methods, by using monkey T cells derived The amplified cDNA was used as a template for the PCR method, and cloned into pcDNA3.1 (+) (Thermo Fisher Scientific) to construct expression vectors cynoCD3ε-pcDNA3.1 and cynoCD3δ-pcDNA3.1.

    1) Modulation of -6-4 monkey antigen gene expression cells    

Lenti-X293T cells were seeded in a 225 cm ² flask so as to be 5.3 × 10 ⁴ cells / cm ² , and cultured in a DMEM medium containing 10% FBS under the conditions of 37 ° C. and 5% CO ₂ overnight. The next day, cynoCD3ε-pcDNA3.1 and cynoCD3δ-pcDNA3.1, or pcDNA3.1-DEST as a control, were each introduced into Lenti-X293T cells using Lipofectamine 2000, and further cultured at 37 ° C and 5% CO ₂ overnight. . The following day, the expression vector was introduced into Lenti-X293T cells and treated with TrypLE Express. After washing the cells with DMEM containing 10% FBS, the cells were adjusted to a concentration of 5 × 10 ⁶ cells / mL in PBS containing 5% FBS. The obtained cell suspension was analyzed by flow cytometry.

    1) Binding analysis of -6-5 to monkey CD3 by flow cytometry    

Flow cytometry was used to further confirm the binding specificity of the antibody produced by the fusion tumor of Example 1) -6-2, which was determined to be a fusion tumor producing antibody against human CD3, to monkey CD3. The Lenti-X293T cell suspension prepared in Example 1) -6-4 was seeded at a rate of 100 μL / well in a 96-well U-bottom microtiter plate, and the supernatant was removed after centrifugation. CytoCD3ε-pcDNA3.1 and cynoCD3δ-pcDNA3.1 were introduced into Lenti-X293T cells and pcDNA3.1-DEST was introduced into Lenti-X293T cells, respectively. The fusion tumor culture supernatant was added to the suspension, and the suspension was left at 4 ° C for 1 hour. After washing once with 5% FBS-containing PBS, an anti-rat IgG FITC conjugate diluted 500-fold with 5% FBS-containing PBS was added and suspended, and left to stand at 4 ° C for 30 minutes. After washing twice with 5% FBS-containing PBS, it was resuspended in 5% FBS-containing PBS containing 2 g / ml 7-aminoradidin D and detected by flow cytometry. Data analysis was performed using Flowjo. After 7-aminoradidin D-positive dead cells were excluded by the gate, a histogram of FITC fluorescence intensity of living cells was made. Choose to generate histograms of fluorescence intensity of pcDNA3.1-DEST introduced into Lenti-X293T cells as a control group, and histograms of cynoCD3ε-pcDNA3.1 and cynoCD3δ-pcDNA3.1 introduced to Lenti-X293T cells toward the strong fluorescence intensity side The fusion tumor of the displaced sample produces a fusion tumor as an antibody that binds to monkey CD3.

    1) -6-6 modulation of human CD3δ gene expression cells    

Lenti-X293T cells were seeded in a 225 cm ² flask to make 5.3 × 10 ⁴ cells / cm ² , and cultured in a DMEM medium containing 10% FBS at 37 ° C. and 5% CO ₂ overnight. The next day, hCD3δ-pcDNA3.1 or pcDNA3.1-DEST as a control was introduced into Lenti-X293T cells using Lipofectamine 2000, and further cultured overnight at 37 ° C and 5% CO ₂ . The next day, the expression vector was introduced into Lenti-X293T cells, treated with TrypLE Express, and the cells were washed with DMEM containing 10% FBS, and then adjusted to a concentration of 5 × 10 ⁶ cells / mL in PBS containing 5% FBS. The obtained cell suspension was analyzed by flow cytometry.

    1) Binding analysis of human CD3δ from 6-7 to flow cytometry    

Furthermore, the specificity of human CD3δ binding antibody produced by a fusion tumor, which was determined to be a fusion tumor produced by an antibody that binds to monkey CD3, was confirmed by flow cytometry in Example 1) -6-5. The Lenti-X293T cell suspension prepared in Example 1) -6-6 was seeded at a rate of 100 L / well in a 96-well U-bottom microplate, and the supernatant was removed after centrifugation. HCD3δ-pcDNA3.1 was introduced into Lenti-X293T cells and pcDNA3.1-DEST was introduced into Lenti-X293T cells, respectively. The fusion tumor culture supernatant was added to the suspension, and the suspension was left at 4 ° C for 1 hour. After washing once with 5% FBS-containing PBS, an anti-rat IgG FITC conjugate diluted 500-fold with 5% FBS-containing PBS was added, suspended, and left to stand at 4 ° C for 30 minutes. After washing twice with 5% FBS-containing PBS, the suspension was resuspended in 5% FBS-containing PBS containing 2 μg / ml 7-aminoradidin D and detected by flow cytometry. Data analysis was performed using Flowjo. After 7-aminoradidin D-positive dead cells were excluded by the gate, a histogram of FITC fluorescence intensity of living cells was made. A histogram of fluorescence intensity of pcDNA3.1-DEST introduced into Lenti-X293T cells as a control was generated, and fusion tumors that produced samples with hCD3δ-pcDNA3.1 introduced into Lenti-X293T cells shifted to the side of strong fluorescence intensity were excluded as Antibodies that bind to human CD3δ produce fusion tumors.

    1) Analysis of conjugated flow cytometry on monkey T cell lines from 6-8    

The binding specificity of the antibodies produced by the antibody-producing fusion tumors of Example 1) -6-7 to monkey T cell lines was further confirmed by flow cytometry. HSC-F (JCRB cell bank, No. JCRB1164), which is a cynomolgus monkey T cell line, was prepared to a concentration of 5 × 10 ⁶ cells / mL in RPMI1640 medium containing FBS, and 100 μL each was inoculated in a 96-well plate. The HSC-F cells from which the supernatant was removed by centrifugation were added to the culture supernatant of the fusion tumor or the rat IgG isotype control antibody that was not excluded in Example 1) -5-7 to produce fusion tumors, and then allowed to stand at 4 ° C. Leave for 1 hour. Then, the supernatant was removed, and the cells in the wells were washed once with 5% FBS-containing PBS, and then anti-rat IgG FITC conjugate diluted 500 times with PBS containing 5% FBS was added and suspended at 4 ° C. Let stand for 1 hour. After washing three times with 5% FBS-containing PBS, it was resuspended in 5% FBS-containing PBS containing 2 μg / ml 7-aminoradiin D and detected by flow cytometry. Data analysis was performed using Flowjo. After the 7-aminoradidin D-positive dead cells were excluded by the gate, a histogram of FITC fluorescence intensity of living cells was made, and a histogram of fluorescence intensity relative to rat IgG isotype control antibody was selected, and FITC fluorescence was selected. The fusion tumor of the sample in which the histogram of the intensity is shifted to the side of the strong fluorescence intensity produces a fusion tumor as an antibody that also binds to the monkey T cell line.

    1) Isotype determination of -7 antibody    

From the rat anti-CD3 antibody-producing fusion tumor obtained in Example 1) -6, C3-, which binds to human and monkey CD3ε, also binds to monkey T cell lines, and is suggested to have high human T cell activation capacity. 147. Identify the antibody isotype. Isotypes were determined using Rat Immunoglobulin Isotyping ELISA Kit (BD Pharmingen). As a result, it was confirmed that the isotype of rat anti-CD3 monoclonal antibody C3-147 was IgG2b and lambda chain.

    (Example 2) Binding of rat anti-CD3 monoclonal antibody (C3-147) to human CD3         2) Preparation of -1 monoclonal antibody from fusion tumor supernatant         2) -1-1 C3-147 fusion tumor culture    

Rat anti-CD3 monoclonal antibody system was purified from the fusion tumor culture supernatant. First, C3-147-producing fusion tumors were propagated to a sufficient amount with ClonaCell-HY Selection Medium E (StemCell Technologies), then 20% Ultra Low IgG FBS (Thermo Fisher Scientific) was added, and the size was 5 μg / mL. Hybridoma SFM (Thermo Fisher Scientific) of Mycin (Thermo Fisher Scientific) was exchanged with a culture medium and cultured for 7 days. This culture supernatant was recovered and sterilized by passing through a 0.22 μm filter (Corning).

    2) -1-2 purification    

From the culture supernatant of the fusion tumor prepared in Example 2) -1-1, the antibody was purified by Protein G affinity chromatography. The antibody was adsorbed on a Protein G column (GE Healthcare Bioscience), and the column was washed with PBS, and then eluted with a 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7). 1M Tris-HCl (pH 9.0) was added to the eluate, and the pH was adjusted to 7.0 to 7.5. The antibody was then replaced with a buffer solution of PBS using Centrifugal UF Filter Device VIVASPIN20 (fractionated molecular weight UF30K, Sartorius). It was concentrated and the antibody concentration was adjusted to 2 mg / mL. Finally, a Minisart-Plus filter (Sartorius) was used as a purified sample.

    2) -2 Obtain rat anti-CD3 antibody (C3-147) binding to human single chain antigen         2) -2-1 Modulation of human CD3εγ single chain antigen    

The amino acid sequence encoding CD3ε or CD3γ is the sequence used in the crystal structure (PDBID: 1SY6) of the OTK3-human CD3εγ single chain antigen complex published in the protein database. The linker system linking the carboxyl terminus of CD3ε and the amino terminus of CD3γ uses the same 26 amines as reported by the reference (Kim, KSet al., (2000) J. Mol. Biol. 302, 899-916) Peptide linker made from amino acid. The gene line encoding the human CD3εγ single-chain antigen shown in Fig. 3 (sequence identification number 4) was synthesized by adding restriction enzyme sites BamHI and Hind III to the 5 'end and 3' end (GENEART). A fragment of approximately 4.8 kb obtained by digesting pQE80L (Qiagen) with restriction enzymes BamHI and Hind III, and a fragment of approximately 0.6 kb obtained by digesting a human CD3εγ single chain antigen gene with BamHI and Hind III, using Ligation High (Toyobo) was combined to produce pQE80L-scCD3εγ, a plastid for coliform expression. The amino acid sequence of the generated scCD3εγ is shown in Fig. 4 (sequence identification number 5). Coliform BL21 (DE3) for expression was transformed with pQE80L-scCD3εγ for expression, and the obtained selected plants were planted in a 1L MagicMedia (Invitrogen) / Ultrayield flask (Thomson) and cultured at 30 ° C with shaking at 250 rpm for 21 days. hour. The cultured bacterial cells were recovered, and in the presence of Tris buffer containing 1% Triton solution, the bacterial cells of the ultrasonic homogenizer were repeatedly broken and frozen and thawed. Finally, the cells were centrifuged at 4 ° C and 15000 rpm for 15 minutes to recover the inclusion bodies. (inclusion body). Refolding from the inclusion body to purification is performed according to the method of reference (Kjer-Nielsen et al. (2004) PNAS vol. 101, no. 20, 7675-7680). However, the anti-CD3 antibody system used for the antibody column is not 2C11 used in the literature, and the mouse anti-CD monoclonal antibody OKT3 (Sgro, Toxicology 105 (1995), 23-29, Orthoclone, Janssen-Cilag) was used.

    2) -2-2 Utilizing the binding of SPR to human CD3εγ single chain antigen    

The antibody-antigen binding system was measured using a Biacore 3000 (GE Healthcare Bioscience), capture method using an antibody as a ligand to capture an immobilized anti-mouse IgG antibody, and measuring the antigen as an analyte. As the antigen, human CD3εγ modulated by 2) -2-1 was used. An anti-mouse IgG antibody (Mouse Antibody Capture Kit, GE Healthcare Bioscience) was covalently bound to the sensor chip CM5 (GE Healthcare Bioscience) at about 11,000 RU by amine coupling. It is similarly fixed to a reference cell. HBS-EP + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P 20) was used as the running buffer. Anti-mouse IgG antibody was immobilized on the wafer, and the antibody was added as a ligand to capture about 1 minute. Then, 100 nM antigen was added at a flow rate of 30 μl / min for 120 seconds, and the binding of the antigen was monitored. As a regeneration solution, 10 mM glycine-HCl pH 1.7 was added at a flow rate of 10 μl / min for 3 minutes. As a result, after C3-147, the combined message after 120 seconds was 34RU.

    2) -3 Confirmation of the antigen-binding site of rat anti-CD3 antibody (C3-147) using SPR    

The method of confirming the antigen-binding site is in accordance with 2) -2-2. As a result, on C3-147, a 34RU binding message was obtained. On the other hand, as a comparative example, the antigen-binding site was similarly confirmed for SP34 (BD Pharmingen), which is a known anti-CD3 antibody. At SP34, no combined message of 34RU was seen. Therefore, the binding site of the CD3εγ surface identified by C3-147 is different from that of SP34.

    (Example 3) Determination of base sequence of cDNA encoding variable region of rat anti-CD3 antibody (C3-147)    

The nucleotide sequence of the cDNA encoding the variable region of the rat anti-CD3 antibody (C3-147) was determined by the following method.

    3) -1 cDNA synthesis    

The rat anti-CD3 antibody (C3-147) was used to produce a fusion tumor cell lysate (50mM Tris-HCl (pH7.5), 250mM LiCl, 5mM EDTA (pH8), 0.5% dodecyl sulfate Li (LiDS), 2.5 mM dithiothreitol (DTT)) was mixed with magnetic beads of Dynabeads mRNA DIRECT Kit (Thermo Fisher Scientific) bound by oligo-dT25 to bind mRNA with magnetic beads. Next, magnetic beads were washed with mRNA washing solution A (10mM Tris-HCl (pH7.5), 0.15M LiCl, 1mM EDTA, 0.1% LiDS, 0.1% TritonX-100) and cDNA synthesis solution (50mM Tris-HCl (pH8.3), 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 0.5 mM dNTP, 0.2% TritonX-100, 1.2 units of RNase inhibitor (Thermo Fisher Scientific) was washed once, and 12 units of SuperScriptIII reverse transcriptase were added (Thermo Fisher Scientific) cDNA synthesis solution was used for cDNA synthesis, and then washed with a 3 'tailing reaction solution (50 mM potassium phosphate, 4 mM MgCl2, 0.5 mM dGTP, 0.2% TritonX-100, 1.2 unit RNase inhibitor) 48 units of terminal transferase were added, and a 3 'tail reaction was carried out by adding a reaction solution of a recombinant (Roche).

    3) Amplification and sequence determination of gene fragments of heavy and light chain variable regions of rat immunoglobulin    

The magnetic beads were washed with TE solution (10mM Tris-HCl (pH7.5), 1mM EDTA, 0.1% TritonX-100), and then subjected to rat immunoglobulin heavy and light chains using 5'-RACE PCR method. Gene gain. That is, the magnetic beads were transferred to a PCR reaction solution (0.2 μM primer, 0.2 mM dNTP, 0.25 unit PrimeSTAR HS DNA Polymerase (TAKARA)), and a reaction at 94 ° C for 30 seconds to 68 ° C for 90 seconds was performed for 35 cycles. The primer set used is as follows.

PCR primer set for heavy chain gene amplification

Sense primer Nhe-polyC-S

5’-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCDN-3 ’FIG. 5 (sequence identification number 50)

The first antisense primer rIgγ-AS1

5’-TCACTGAGCTGGTGAGAGTGTAGAGCCC-3 ’FIG. 6 (sequence identification number 51)

Second antisense primer rIgγ-AS2

5’-TCACCGAGCTGCTGAGGGTGTAGAGCCC-3 ’FIG. 7 (sequence identification number 52)

PCR primer set for light chain gene amplification

Sense primer Nhe-polyC-S2

5’-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCDCCD-3 ’FIG. 8 (sequence identification number 53)

The first antisense primer rIgL-AS1

5’-TTCCACATCACTCGGGTAGAAATCAG-3 ’FIG. 9 (sequence identification number 54)

Second antisense primer rIgγ-AS2

5’-TAACACCAGGGTAGAAATCTGTCACCAT-3 ’FIG. 10 (sequence identification number 55)

Sequence analysis of the base sequence was performed on the fragments amplified by the PCR reaction. The primers used are as follows.

Sense primer rIgγ-seq for heavy chain sequencing

5’-2TGGCTCAGGGAAATAGCC-3 ’FIG. 11 (sequence identification number 56),

Antisense primer rIgL-seq1 for light chain sequencing

5’-TCCCTGGAGCTCCTCAGT-3 ’FIG. 12 (sequence identification number 57)

Antisense primer rIgL-seq2 for light chain sequencing

5’-GCCTTGTCAGTCTTGAGC-3 ’FIG. 13 (sequence identification number 58)

The sequence analysis was performed using a gene sequence analysis device ("ABI PRISM 3700 DNA Analyzer; Applied Biosystems" or "Applied Biosystems 3730xl Analyzer; Applied Biosystems"). The sequencing reaction was performed using Dye Terminator Cycle Sequencing System with AmpliTaq DNA polymerase (Life Technologies Company) and GeneAmp 9700 (Applied Biosystems).

The base sequence of the C3-147 heavy chain variable region determined by sequence analysis is shown in FIG. 14 (sequence identification number 6), the amino acid sequence is shown in FIG. 15 (sequence identification number 7), and C3-147 The base sequence of the chain variable region is described in FIG. 16 (sequence identification number 8), and the amino acid sequence is described in FIG. 17 (sequence identification number 9).

    (Example 4) Preparation of rat anti-CD3 scFv (C3E-7000) and its humanized form (C3E-7034)         4) -1 Preparation of rat anti-CD3 antibody (C3-147) scFv         4) -1-1 Construction of rat antibody CD3 scFv expression vector (pC3E-7000)    

A sense oligo of a DNA fragment having a 15-base additional sequence before and after the DNA sequence encoding the linker inserted between the C3-147 heavy chain variable region (VH) and the light chain variable region (VL) Nucleotide Figure 18 (sequence identification number 10), and Figure 19 (Sequence ID 11) for synthesizing the antisense strand oligonucleotide (Sigma Aldrich, custom oligonucleotide synthesis service), adjusted to 100 pmol / μL Then, each 20 μL was mixed, and the two were annealed by being left to stand between 96 ° C for 10 minutes, 70 ° C for 2 minutes, 60 ° C for 2 minutes, 40 ° C for 2 minutes, and 30 ° C for 2 minutes, and inserted between VH and VL. Linker fragment. Secondly, the method of attaching human IgG heavy chain message sequences to the vector backbone derived from the animal cell expression vector pcDNA-3.3TOPO (Thermo Scientific) uses the In-Fusion HD selection kit to make DNA fragments amplified by PCR, A DNA fragment amplified by PCR of VH of rat anti-CD3 antibody C3-147 shown in FIG. 14 (sequence identification number 6), a linker fragment inserted between VH and VL, and FIG. 16 (sequence identification number 8) The C3-147 shown in the VL-containing region binds the DNA sequence encoding the FLAG-His tag to the carboxyl-terminated PCR amplified DNA fragment to produce a nucleotide sequence containing the nucleotide sequence of FIG. 20 (SEQ ID NO: 14). ScFv expression vector pC3E-7000 on ORF.

    4) -1-2 rat anti-CD3 scFv (C3E-7000) performance and purification    

Expi293F cells (Thermo Scientific) were subcultured and cultured according to the manual. Expi293F cells in the logarithmic proliferation phase are introduced into the scFv expression vector described above for temporary expression, and filtered for purification. Purification was performed in a two-step process using Ni affinity chromatography using His Trap excel (GE Healthcare) and colloid filtration using Superdex 200increase (GE Healthcare). A peak corresponding to the molecular weight of the scFv monomer was recovered as a purified protein sample. . For purification, all steps were performed at 4 ° C using an AKTA chromatography system. The purified protein buffer was HBSor (25 mM histidine / 5% sorbitol, pH 5.0). The purified protein sample is supplied to the SEC for analysis, and the purity and concentration are determined and then used for various analyses. The amino acid sequence of C3E-7000 is shown in Figure 21 (SEQ ID NO: 15).

    4) -2 Humanization of rat anti-CD3 scFv (C3E-7000)         4) -2-1 Humanized Design of Anti-CD3 Antibodies    

The molecular simulation of the variable region of the rat antibody was performed in accordance with a well-known method (homology modeling) (Methods in Enzymology, 203, 121-153, (1991)) using a commercially available protein stereostructure analysis program Discovery Studio3 .5 (Dassault Systems).

Humanization was performed according to a generally known method as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The receptor resistance system is based on the subgroup of humans identified by KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) The amino acid identity of the sequence or the framework region of the Germline sequence or the predicted score of immunogenicity and physical properties are selected. In addition, using the three-dimensional structure model constructed by the above-mentioned method, and referring to the benchmark proposed by Queen et al. (PNAS (1989) 86, 10029-10033), the mutation was selected to be returned.

    4) Design of humanized amino acid sequence of 2-2 C3E-7000    

Based on the method described in Example 4) -2-1, γ3 and λ6 of human subgroups and consensus sequences were used as receptors to design the amino acid sequence of C3E-7034, which is a humanized form of C3E-7000. In the amino acid sequence of C3-147VH shown in FIG. 15 (sequence identification number 7), the arginine at the 16th position of the amino acid number is replaced by the glycine, and the alanine at the 17th position of the amino acid number is accompanied. Substitution with serine, lysine at position 19, arginine, valine at position 23, valine at position 23, alanine, and valine at position 24 Designed C3E-7034VH amino acid sequence of C3E-7034VH, which is substituted with alanine, serine at amino acid number 88 is replaced with alanine, and threonine at amino acid number 93 is replaced with valine. , Described in Figure 22 (serial identification number 16).

In the amino acid sequence of C3-147VL shown in FIG. 17 (sequence identification number 9), the amino acid number 1 in the amino acid number is replaced with aspartic acid and the amino acid number 3 Valine was replaced with methionine, aspartic acid at position 8 was replaced by histamine, threonine at position 12 was replaced by glutamic acid, amine The aspartic acid at position 13 is replaced by serine, the leucine at position 14 is replaced by proline, and the threonine at position 16 is replaced by amine Acid, glutamic acid at amino acid number 19 is replaced by threonine, leucine at amino acid number 20 is replaced by isoleucine, and arginine at amino acid number 43 Substituted for serine, serine for amino acid number 75, serine, aspartic acid for amino acid number 79, serine, amino acid number The amino acid sequence of C3E-7034VL designed by substitution of valine at position 81 with leucine and glutamic acid at position 82 with lysine is shown in Figure 23 (SEQ ID NO: 17) ). The CDR sequences of C3E-7000 and C3E-7034 in the CDR definition of IMGT are shown in FIG. 24 (sequence identification number 26), CDR-H2 in FIG. 25 (sequence identification number 27), and CDR-H3. It is shown in FIG. 26 (sequence identification number 28), CDR-L1 is shown in FIG. 27 (sequence identification number 29), CDR-L2 is shown in FIG. 28 (sequence identification number 30), and CDR-L3 is shown in FIG. 29 (sequence identification number) 31).

    4) -2-3 Changes of humanized anti-CD3 scFvC3E-7034    

In order to produce a mutant that maintains crossover to monkey CD3ε and has different binding activity and cytotoxic activity, the VL of C3E-7034 was framed in the same manner as described in Example 4) -2-1. The amino acid in the region was designed as a variant in which the VL of scFv (4 variant sequences of A2S, S8P, V13A, and F80L were placed in IGLV1-40 * 01).

    4) Amino acid sequence design of 2-3-1 C3E-7035    

The amino acid sequence of C3E-7035 designed to be a variant of C3E-7034. In the light chain of C3E-7034 shown in FIG. 23 (SEQ ID NO: 17), the aspartic acid in the first position of the amino acid number of the variable region is replaced with the glutamic acid, and the amino acid number in the Substitution of phenylalanine at position 2 with alanine, methionine at position 3 with valine, histamine at position 8 with serine, and serine at position 8 Replacement of glutamic acid at position 12 with glycine, serine at position 13 with valine, and lysine at position 16 with glutamic acid and amino acid The threonine at number 17 is replaced by arginine, the histamine at number 40 is replaced by leucine, and the glutamic acid at position 41 is replaced by proline and amine Substitution of serine at position 43 to threonine, substitution of serine at position 44 to alanine, substitution of threonine at position 46 to lysine, amine Substitution of threonine at position 47 with leucine, isoleucine at position 48 with leucine, and aspartic acid at position 57 with serine Amine Substitution of serine at number 60 with proline, substitution of isoleucine at position 67 with lysine, deletion of aspartic acid at position 68, deletion of amino acid Substituted arginine at position 69, serine at position 71 at glycine, glycine at position 72, lysine at position 72, threonine at position 77 The threonine was replaced by alanine, the serine at amino acid number 79 was replaced by threonine, the aspartic acid at amino acid number 80 was replaced by glycine, and the amino acid number was Substitution of leucine at position 81 with phenylalanine, replacement of lysine at position 82 with glutamic acid, threonine at position 83 with alanine and alanine The amino acid sequence of the designed C3E-7035 light chain in which the phenylalanine at position 90 is replaced with tyrosine is shown in Figure 30 (sequence identification number 20). Methionine and The full-length sequence of C3E-7035 of alanine is shown in FIG. 31 (SEQ ID NO: 22).

    4) -2-3-2 C3E-7036 amino acid sequence design    

The amino acid sequence of C3E-7036 designed to be a variant of C3E-7034. The histidine at position 8 at the amino acid number of the variable region in the light chain of C3E-7034 shown in FIG. 23 (SEQ ID NO: 17) is replaced with serine and glutamine at position 12 Acid substitution with glycine, serine at amino acid number 13 with valine, lysine at amino acid number 16 with glutamic acid, amino acid with number 17 Substituting threonine for arginine, amino acid number 23 for lysine, threonine, amino acid number 24 for arginine, glycine, amino acid number 40 The histidine was replaced with leucine, the glutamic acid at position 41 was replaced by proline, the serine at position 43 was replaced by threonine, and the amine was numbered 44 Substituted serine for alanine, threonine at amino acid number 46 for lysine, threonine at amino acid number 47 for leucine, and amino acid number 48 Isoleucine at position I is replaced by leucine, aspartic acid at position 57 is replaced by serine, serine at position 60 is replaced by proline and amino acid Number 67 Isoleucine was replaced with lysine, aspartic acid at position 68 was deleted, arginine at position 69 was deleted, serine at position 71 was replaced by Glycine, lysine at amino acid number 72 is replaced with threonine, lysine at amino acid number 77 is replaced with alanine, and serine at amino acid number 79 is replaced with Threonine, aspartic acid at position 80 is replaced with glycine, leucine at position 81 is replaced by phenylalanine, and lysine at position 82 C3E-7036 amino acid of light chain design which is substituted with glutamic acid, threonine at amino acid number 83 is substituted with alanine, and phenylalanine at amino acid number 90 is substituted with tyrosine The sequence is described in FIG. 32 (sequence identification number 23), and the full-length amino acid sequence of C3E-7036 is described in FIG. 33 (sequence identification number 25).

    4) -2-3-3 amino acid sequence design of CDR variants    

For the purpose of removing the deamination site existing in CDRH2 (Figure 25, sequence identification number 27) of C3E-7034, the amine group of the heavy chain variable region of C3E-7034 shown in Figure 22 (SEQ ID NO: 16) was designed. The aspartic acid at position 53 of the acid number is C3E-7078 with aspartic acid and C3E-7079 with serine. In addition, the aspartic acid at position 53 of the amino acid number of the heavy chain variable region of C3E-7036 was designed to be substituted with C3E-7085 of arginine. The full-length amino acid sequence of C3E-7078 is described in Figure _68 (sequence identification number 60), the full-length amino acid sequence of C3E-7079 is described in Figure 70 (sequence identification number 62), and the full-length amino acid sequence of C3E-7085 It is described in FIG. 72 (serial identification number 64).

Furthermore, for the purpose of reducing the affinity of human CD3 of C3E-7078, the aspartic acid at position 52 of the amino acid number of the light chain variable region of C3E-7078 was designed to be substituted with glycine C3E-7086, C3E-7087 with glutamic acid, C3E-7088 with aspartic acid, C3E-7089 with serine, and C3E-7090 with alanine. Similarly, for the purpose of reducing the human CD3 affinity of C3E-7079, C3E-7091 was designed to replace aspartic acid at position 52 of the amino acid number of the light chain variable region of C3E-7079 with glycine. , C3E-7092 with glutamic acid, C3E-7093 with aspartic acid, C3E-7094 with serine, and C3E-7095 with alanine. The full-length amino acid sequence of C3E-7086 is described in FIG. 74 (sequence identification number 66), the full-length amino acid sequence of C3E-7087 is described in FIG. 76 (sequence identification number 68), and the full-length amino acid sequence of C3E-7088 The full-length amino acid sequence of C3E-7089 is described in FIG. 78 (sequence identification number 70), and the full-length amino acid sequence of C3E-7090 is described in FIG. 82 (sequence identification number 74). The full-length amino acid sequence of C3E-7091 is described in FIG. 84 (sequence identification number 76), the full-length amino acid sequence of C3E-7092 is described in FIG. 86 (sequence identification number 78), and the full-length amino acid sequence of C3E-7093 The full-length amino acid sequence of C3E-7094 is described in FIG. 88 (sequence identification number 80), and the full-length amino acid sequence of C3E-7095 is described in FIG. 92 (sequence identification number 84). .

    4) -3 Production of humanized anti-CD3 scFv (C3E-7034, C3E-7035, C3E-7036)         4) -3-1 Construction of humanized anti-CD3 scFv (C3E-7034) expression vector pC3E-7034    

The carboxy terminus of the C3E-7034 heavy chain shown in FIG. 22 (SEQ ID NO: 16) was synthesized by interposing 15 amino acid flexible linkers, and the C3E- A 70F light chain region-linked scFvDNA sequence and a DNA fragment containing an additional sequence of 15 bases before and after (GENEART). Using this as a template, C3E-7034 and the region containing the additional sequence before and after were amplified by the PCR method to obtain an inserted DNA fragment. Furthermore, the expression vector pC3E-7000 prepared in Example 4) -1-1 was used as a template, and the vector region excluding the scFv region was amplified by the PCR method to obtain a vector fragment. The respective DNA fragments were combined using the In-Fusion HD selection kit (CLONTECH), and a humanized anti-CD3 scFv expression vector pC3E-7034 including the nucleotide sequence of FIG. 34 (SEQ ID NO: 18) in the ORF was produced.

    4) Construction of 3-2 humanized anti-CD3 scFv (C3E-7035) expression vector pC3E-7035    

A flexible linker composed of 17 amino acids was synthesized at the carboxy terminus of the C3E-7034 heavy chain shown in FIG. 22 (SEQ ID NO: 16), and the C3E- shown in FIG. 30 (SEQ ID NO: 20) was synthesized. 7035 light chain-linked scFvDNA sequence and a DNA fragment containing an additional sequence of 15 bases before and after (GENEART). In the same manner as in Example 4) -3-1, a C3E-7035 expression vector containing the nucleotide sequence of FIG. 35 (sequence identification number 21) in the ORF was constructed. The obtained expression vector was named "pC3E-7035".

    4) Construction of 3-2 humanized anti-CD3 scFv (C3E-7036) expression vector pC3E-7036    

The C3E-7034 heavy chain shown in FIG. 22 (SEQ ID NO: 16) was synthesized by carboxyl terminus with 15 flexible linkers interposed therebetween to link the C3E-7036 light chain shown in FIG. 32 (SEQ ID NO: 23). ScFvDNA sequence and a DNA fragment containing an additional sequence of 15 bases before and after (GENEART). In the same manner as in Example 4) -3-1, a C3E-7036 expression vector containing the nucleotide sequence of FIG. 36 (SEQ ID NO: 24) in the ORF was constructed. The obtained expression vector was named "pC3E-7036".

    4) -3-4CDR changes the expression vector construction of humanized antibody CD3 scFv    

Using the nucleotide sequence of C3E-7034 shown in FIG. 34 (sequence identification number 18) in pC3E-7034 of ORF as a template, the base sequence shown in FIGS. 93 and 94 (sequence identification numbers 85 and 86) was used. The primers were used to introduce site-specific mutations using PCR to create a nucleus of C3E-7078 containing aspartic acid at position 53 of the heavy chain variable region of C3E-7034. The nucleotide sequence is in the C3E-7078 expression vector of ORF. The obtained expression vector was named "pC3E-7078". Similarly, pC3E-7034 was used as a template, and Figs. 95 and 96 (sequence identification numbers 87 and 88) were used as primers, and site-specific mutations using PCR were introduced to prepare an amine containing a heavy chain variable region of C3E-7034. The nucleotide sequence of C3E-7079 of aspartic acid with serine at position 53 of the amino acid number is substituted in the C3E-7079 expression vector of ORF. The obtained expression vector was named "pC3E-7079". Similarly, pC3E-7036 was used as a template, and Figs. 93 and 94 (sequence identification numbers 85 and 86) were used as primers, and site-specific mutations using PCR were introduced to prepare an amine containing a heavy chain variable region of C3E-7036. The nucleotide sequence of C3E-7085 of aspartic acid with arginine at position 53 of the amino acid number is substituted in the C3E-7085 expression vector of ORF. The obtained expression vector was named "pC3E-7085".

C3E-7086 replacing aspartic acid with glycine at position 52 of the amino acid number of the light chain variable region of C3E-7078, C3E-7087 with glutamic acid, aspartic acid Each nucleotide sequence of substituted C3E-7088, serine substituted C3E-7089, alanine substituted C3E-7090 in the expression vector of ORF, and amine containing the light chain variable region of C3E-7079 C3E-7091 with aspartic acid replaced with glycine at position 52 of the amino acid number, C3E-7092 with glutamic acid, C3E-7093 with aspartic acid, and serine Each nucleotide sequence of C3E-7094 and alanine-substituted C3E-7095 on the ORF expression vector was also prepared in the same manner. The created vector name, template, and list of primers are listed in Table 1 together, and the list of primers is listed in FIG. 105.

    4) -3-5 Performance of humanized anti-CD3 scFv‧purification    

The expression and purification of C3E-7034, C3E-7035, and C3E-7036 were performed in the same manner as in Example 4) -1-2.

    4) -3-6 CDR changes the performance of humanized anti-CD3 scFv‧Purification    

C3E-7078, C3E-7079, C3E-7085, C3E-7086, C3E-7087, C3E-7088, C3E-7089, C3E-7090, C3E-7091, C3E-7092, C3E-7093, C3E-7094, C3E- The expression and purification of each CDR variant of 7095 was performed in the same manner as in Example 4) -1-2.

    (Example 5) Crystal structure analysis of humanized anti-CD3 scFv (C3E-7034)         5) -1 Modulation of humanized anti-CD3 scFv (C3E-7034) -human CD3εγ single chain antigen complex    

The CD3εγ produced in Example 2) -2-1 and the C3E-7034 produced in Example 4) -3-1 were mixed at a molar ratio of 1: 2, and Amicon Ultra15 MWCO 10kilo (Millipore) was used to replace the buffer solution. 10 mM Tris HCl (pH 7.5), 50 mM NaCl, and concentrated to 3.5 mg / mL. This was purified by colloidal filtration chromatography using Superdex200 10 / 300GL (GE Healthcare), and the complex fraction was concentrated to approximately 4.0 mg / mL using Amicon Ultra 15MWCO 10kilo (Millipore).

    5) -2 crystallization    

The obtained complex of CD3εγ and C3E-7034 was crystallized by a vapor diffusion method. A precipitant solution (0.1M MES monohydrate (pH 6.5), 1.6M ammonium sulfate, 10% v / v 1,4-dichloromethane, 0.5 μL of protein solution) Alkane) solution was collected in a closed container containing 0.05 mL of a precipitant solution so that the two solutions were not in contact, and left to stand at 25 ° C. One month later, rod-shaped crystals of 0.1 mm × 0.05 mm × 0.05 mm were obtained.

    5) -3 X-ray crystal structure analysis and epitope identification    

The obtained crystals were immersed in Perfluoropolyether PFO-X175 / 08 (Hampton Research), and then frozen with liquid nitrogen. X-ray diffraction data were collected with a beam line BL41XU (SPring-8, Hyogo). From the diffraction image obtained, the software imosflm (CCP4: Collaborative Computational Project No. 4) was used to digitize the diffraction intensity to obtain the crystal structure factor. The crystal is a hexagonal system, the space group is P62, and the unit lattice system of the crystal is a = 193.54Å, b = 193.54Å, and c = 43.88Å.

Using the obtained structural factors and the three-dimensional structural coordinates of the homology simulation, a molecular replacement method is performed to determine the phase. The software uses phaser (CCP4: Collaborative Computational Project No. 4) for calculation. The crystal system contains one complex in an asymmetric unit.

Software Refmac5 (CCP4: Collaborative Computational Project No. 4) was used to refine the structure, and software coot was used to correct the model. Repeat this operation and decompose the energy at 3.3Å to obtain the final R value of 22.1% and the free R value of 27.0%. The final model is the amino acid residues 1-108 of the light chain region of C3E-7034 (Figure 23, sequence identification number 17), and the amino group of the heavy chain region of C3E-7034 (Figure 22, sequence identification number 16). Acid residues 1-118, amino acid residues 33-67 and 71-118 in the CD3ε region (Figure 1, sequence identification number 1), and amino acid residues in the CD3γ region (Figure 37, sequence identification number 3) 23-103. Amino acid residues 68-70 in the CD3ε region (Figure 1, sequence identification number 1), and the amino terminal region (amino acid residue 1), linker in C3E-7034 (Figure 38, sequence identification number 19) Parts (amino acid residues 120-134) and carboxyl terminal regions (amino acid residues 243-269) were not modeled because the respective electron densities were unknown. The ribbon model and surface of the entire complex are shown in FIG. 39.

The interaction of CD3ε with the light and heavy chains of C3E-7034 is shown in Figure 40. Group A is a diagram of the amino acid residues of CD3ε of C3E-7034 within a distance of less than 4 Å from the variable region of the light chain by a thick stick model, and the amino acid residues other than that by a thin stick model. Ser55, Glu56, Arg101, Gly102, Ser103, Lys104, and Pro105 C3E-7034 within the squares of the figure indicate the amino acid of CD3ε within a distance of 4 Å from the light chain variable region. Residues, each amino acid number corresponds to the sequence identification number 1 of the sequence listing. Group B is a diagram of the amino acid residues of CD3ε of CD3ε with a distance of less than 4 Å from the variable region of the heavy chain of C3E-7034 using a thick stick model, and the amino acids other than this are shown by a thin stick model. In the figure, the residue names and residue numbers in the squares Ser55, Glu56, Leu58, Trp59, Asn65, Ile66, Ser77, Asp78, and Arg101 are the amino acids of CD3ε, and each amino acid number corresponds to the sequence table The serial identification number 1. The distance from C3E-70344Å is interpreted as the amino acid residues of the epitope of CD3ε of C3E-7034 as follows: Ser55, Glu56, Leu58, Trp59, Asn65, Ile66, Ser77, Asp78, Arg101, Gly102, Ser103 , Lys104, and Pro105.

Among the epitopes of CD3ε to C3E-7034, Arg101, Gly102, Ser103, Lys104, and Pro105 are also epitope residues of CD3ε common to OKT3 and UCHT1 (Kjer-Nielsen et al., PNAS 101 (2004) , P.7675-80; Arnett et al., PNAS 101 (2004), p. 16268-73). In addition, C3E-7034 proved to have no interaction with amino acid numbers 22 to 48 in the sequence identification number 1 corresponding to the epitope of an anti-CD3 antibody including I2C, H2C, and the like described in WO2008 / 119565A2. In Figure 41, the CD3ε sequence presents interacting residues. The message sequence of CD3ε is shown in italics, and the amino acid within 4Å of C3E-7034 is underlined.

    (Example 6) Production of humanized OKT3 scFv         6) -1 Construction of OKT3 scFv expression vector pC3E-3000    

In the same manner as in Example 4) -1-1, a mouse anti-CD3 monoclonal antibody OKT3 (Sgro, Toxicology 105 (1995), 23-29, Orthoclone, Janssen-Cilag) scFv was prepared and introduced into pcDNA3. .3 animal cell expression vector. The obtained expression vector was named "pC3E-3000".

    6) -2 Design of humanized amino acid sequence of OKT3 scFv (C3E-3000)    

Based on the method described in Example 4) -2-1, using the human subgroup ‧ consensus sequences of gamma1 and kappa4 as receptors, the amino acid sequence of C3E-3007 of the humanized body of OKT3 was designed. In addition, considering the effect on the immunogenicity score and physical properties, the amino acid of kappa1 was introduced in some places. In the heavy chain of OKT3 shown in FIG. 42 (SEQ ID NO: 36), the glutamic acid at position 5 of the amino acid number accompanying the variable region is replaced with valine and white at position 11 of the amino acid number. Replacement of amino acid with serine, alanine with amino acid number 12 is replaced with lysine, arginine with amino acid number 13 is replaced with lysine, with amino acid number 20 Substituting thiamin for valine, amino acid number 38 for lysine, arginine, amino acid number 40 for arginine, alanine, and amino acid number 48 for arginine Isoleucine is replaced by methionine, lysine is replaced by arginine at position 67, lysine is replaced by valine, and lysine is placed at position 68 Substituted leucine at iso position to isoleucine, substituted threonine at position 72 of amino acid to alanine, substituted serine at position 76 of amino acid to threonine, amino acid numbered Replacement of glutamic acid at position 82 with glutamic acid, threonine at position 87 with arginine, serine at position 91 with uramine and amino acid Make up The amino acid sequence of the C3E-3007 heavy chain of the designed C3E-3007 heavy chain was replaced by leucine at position 114 and leucine at position 115. No. 38).

In the light chain variable region of OKT3 shown in FIG. 44 (SEQ ID NO: 37), the valamic acid at position 3 of the amino acid number is replaced with glutamic acid, and white at position 4 of the amino acid number Substitute amino acid for methionine, alanine at amino acid number 9 for serine, isoleucine at amino acid number 10 for serine, amino acid number 11 The methionine was replaced by leucine, the serine at position 12 was replaced by alanine, the alanine at position 13 was replaced by valine, and the amino acid was placed at position 15 Proline is replaced by leucine, lysine is replaced by arginine at position 18, valine is replaced by alanine at position 19, and valine is replaced by alanine at position 21 Methionine was replaced by isoleucine, serine at position 39 of the amino acid was replaced by proline, threonine at position 41 of the amino acid was replaced by lysine, and amino acid numbered Substitution of serine at position 42 with alanine, substitution of alanine at amino acid number 59 with aspartic acid, substitution of histamine at amino acid number 60 with arginine, amino acid number Substitution of arginine at position 62 with serine, serine at position 69 with aspartic acid, tyrosine at position 70 with phenylalanine and amino acid The serine at position 71 is replaced by threonine, the glycine at position 76 is replaced by serine, and the methionine at position 77 is replaced by leucine and amine The glutamic acid at position 78 is replaced by glutamic acid, the alanine at position 82 is replaced by valine, the serine at position 99 is replaced by glutamic acid, The amino acid sequence of the C3E-3007 light chain of the designed C3E-3007 light chain in which the leucine at position 103 of the amino acid number is substituted with valine is shown in Figure 45 (SEQ ID NO: 39).

    6) -3 Construction of humanized OKT3 scFv (C3E-3007) expression vector pC3E-3007    

The carboxy terminus of the C3E-3007 heavy chain shown in FIG. 43 (SEQ ID NO: 38) is synthesized through the 15 amino acid flexible linkers and contains the C3E-3007 shown in FIG. 45 (SEQ ID NO: 39). The scFvDNA sequence linked to the strand region and a DNA fragment containing an additional sequence of 15 bases before and after (GENEART). In the same manner as in Example 4) -3-1, a C3E-3007 expression vector containing the nucleotide sequence of FIG. 46 (SEQ ID NO: 34) in the ORF was constructed. The obtained expression vector was named "pC3E-3007".

    6) -4 Performance of humanized OKT3 scFv (C3E-3007) ‧Purification    

Performance and purification of C3E-3007 was performed in the same manner as in Example 4) -1-2. The amino acid sequence of C3E-3007 is shown in Figure 47 (SEQ ID NO: 35).

    (Example 7) In vitro activity of humanized anti-CD3 scFv         7) -1 Study on the binding of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) to human CD3         7) -1-1 Humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) and its binding to human CD3 using flow cytometry    

5% FBS in PBS containing commercially available human PBMC (CTL) was prepared to an appropriate concentration, and LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) and anti-CD19 antibody (Beckman Coulter) were added. Let stand at 4 ° C for 30 minutes. After washing twice with 5% FBS-containing PBS, the concentration was adjusted to 1 × 10 ⁶ cells / mL with 5% FBS-containing PBS, and 100 μL / well was inoculated into a 96-well U-bottom microplate, which was removed after centrifugation. Serum. 100 μL / well of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, and C3E-7036) diluted in PBS containing 5% FBS was added and left at 4 ° C for 60 minutes. After washing twice with 5% FBS-containing PBS, 30 μL / well of Penta-His Alexa Fluor 488 (QIAGEN) diluted with 5% FBS-containing PBS was added, and left to stand at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS, it was resuspended in 5% FBS-containing PBS and detected by a flow cytometer (FACSCanto (trademark) II: BD). Data analysis was performed by Flowjo (Treestar), and the average fluorescence intensity (MFI) of Alexa Fluor 488 was calculated from the fraction of dead cells and CD19-positive cells. The MFI value of the scFv-added sample was subtracted from the MFI value of the antibody-free sample. , Calculate the relative value of the MFI value (rMFI). As shown in Figure 48, humanized anti-CD3 scFv was shown to bind to human CD3.

    7) -1-2 Humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) and SPR binding to human CD3    

The affinity of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) for CD3 was determined using the surface plasmon resonance method using BIAcore T-200 (GE Healthcare). Five scFvs of different concentrations were made to flow into CD3 fixed on the sensor chip, Rmax was estimated from the obtained reaction, and 1/2 of the antibody concentration was taken as the dissociation constant of scFv to CD3. As a result, the dissociation constants of the scFv to CD3 were 400, 4.5, 22, and 25 nM, respectively.

    7) -2 Study on the binding of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7036) to cynomolgus CD3         7) -2-1 modulation of cynomolgus monkey PBMC    

SepMate (STEMCELL) and Lymphocyte Separation Solution (Nacalai Tesque) were used to feed the blood of crab macaques, and PBMCs were collected according to the usual method.

    7) Binding of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) to cynomolgus monkey CD3 using flow cytometry    

The cynomolgus cynomolgus monkey PBMC obtained in Example 7) -2-1 was adjusted to an appropriate concentration in PBS containing 5% FBS, and stained and analyzed in the same manner as in Example 7) -1-1. As shown in Figure 49, humanized anti-CD3 scFvs (C3E-7034, C3E-7035, C3E-7036) were shown to bind to cynomolgus monkey CD3.

    7) -3 Humanized anti-CD3 scFv (C3E-3007, C3E-7034) T cell activation    

Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats from random donors using a concentration gradient density separation method using Lympholyte-H (Cedarlane). LR10 (FBS RPMI1640 containing 10% ultra low IgG (Thermo Fisher Scientific)) was used to dilute 100nM humanized anti-CD3 scFv (C3E-3007, C3E-7034) with the same concentration of anti-His antibody (Qiagen) ) Mix the same amount. Human or monkey PBMCs were prepared into 2 × 10 ⁵ cells with LR10 and mixed in the same amount with humanized anti-CD3 scFv mixed with mixed anti-His antibody in a 96-well U-bottomed microtiter plate, 24 hours at 37 ° C, 5% CO ₂ Culture. After completion of the reaction, centrifuge and add sorter buffer (HBSS (-) (Thermo Fisher Scientific), 0.1% BSA (Sigma-Aldrich), 0.1% sodium azide (Sigma-Aldrich)) After centrifugation, LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) was added to the cells, and the cells were allowed to stand at 4 ° C for 20 minutes. After washing with the screening buffer, the PE-labeled anti-CD69 antibody (Becton, Dickinson) and FITC-labeled anti-CD8 antibody (Becton, Dickinson) diluted with the sorting buffer were added, and left at 4 ° C for 20 minutes. After washing with sorting buffer, it was resuspended in PBS (Wako Pure Chemical Industries, Ltd.) containing 1% paraformaldehyde and detected by a flow cytometer (FACSCanto II: Becton, Dickinson). Data analysis was performed with Flowjo (Treestar). From the fraction of dead cells, the ratio of CD8 high performance and PE high performance fractions was calculated as the percentage from the parent group (% of parents,% of parents). As shown in FIG. 50, the humanized anti-CD3 scFv line shows activation of human and monkey CD8 high-performance cells.

    7) -4 CDR changes in humanized anti-CD3 scFv binding to human and cynomolgus CD3 by flow cytometry    

The human PBMC obtained in Example 7) -1-1 and the cynomolgus cynomolgus monkey PBMC obtained in 7) -2-1 were adjusted to an appropriate concentration in PBS containing 5% FBS to be the same as in Example 7) -1-1. The method is dyed and analyzed. As shown in Fig. 106, it was confirmed that the CDR altered the binding of humanized anti-CD3 scFv to human and monkey CD3.

    (Example 8) Preparation of humanized anti-TROP2 scFv         8) -1 Construction of HT1-11 scFv expression vector pHT1-11scFv    

A DNA fragment (GENEART) containing a DNA sequence encoding the amino acid of the HT1-11 scFv shown in Fig. 51 (sequence identification number 41) was synthesized. Using the In-Fusion HD PCR selection kit (Clontech), a DNA fragment synthesized by inserting a vector derived from pcDNA-3.3TOPO (Thermo Scientific) was used to construct a nucleus shown in FIG. 52 (SEQ ID NO: 40). The nucleotide sequence is in the humanized anti-TROP2 scFv expression vector pHT1-11 scFv of ORF.

    8) -2 Performance and purification of HT1-11 scFv    

The expression and purification of HT1-11scFv were performed in the same manner as in Example 4) -1-2.

    (Example 9) Evaluation of binding to human TROP2 by flow cytometry using humanized anti-TROP2 scFv (HT1-11scFv)    

The pharyngeal flat epithelial cancer cell line FaDu (ATCC) and the pancreatic cancer cell line HPAF-II (ATCC) were adjusted to an appropriate concentration with 5% FBS in PBS, and LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit was added. Let stand for 30 minutes at ℃. After washing twice with 5% FBS-containing PBS, the solution was adjusted to a concentration of 1 × 10 ⁶ cells / mL with 5% FBS-containing PBS, and 100 μL / well was inoculated into a 96-well U-bottomed microtiter disk. Serum. Humanized anti-TROP2 scFv (HT1-11 scFv) diluted in PBS containing 5% FBS was added at 100 µL / well, and left at 4 ° C for 60 minutes. After washing twice with 5% FBS-containing PBS, 30 μL / well of Penta-His Alexa Fluor 488 diluted with 5% FBS-containing PBS was added, and left at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS, it was resuspended in 5% FBS-containing PBS and detected by a flow cytometer (FACSCanto (trademark) II). Data analysis was performed with Flowjo. The average fluorescence intensity (MFI) of the Alexa Fluor 488-removed fraction was calculated. The MFI value of the scFv-added sample was subtracted from the MFI value of the antibody-free sample to calculate the relative value of the MFI value rMFI). As shown in Fig. 53, humanized anti-TROP2 scFv was shown to bind to human TROP2.

    (Example 10) Preparation of anti-TROP2-CD3 bispecific molecule         10) -1 Construction of anti-TROP2-CD3 bispecific molecular expression vector         10) -1-1 Construction of HT1-11scFv / C3E-7034 bispecific molecule (T2C-0001) expression vector    

Using the pHT1-11scFv prepared in Example 8) -1 as a template, a linker between the HT1-11scFv and the human antibody heavy chain message sequence designed at the 5 'side and the scFv at the 3' side was used. Primers were subjected to PCR to obtain inserted DNA fragments. Using the expression vector pC3E-7034 prepared in 4) -3-1 as a template, PCR was performed using the message sequence and primers encoding the amine terminal sequence of the anti-CD3 scFv to obtain a vector containing the entire region of the vector containing the anti-CD3 scFv. Vector DNA fragment. The respective DNA fragments were combined using the In-Fusion HD selection kit (CLONTECH) to produce an anti-TROP2-CD3 bispecific molecular expression vector containing the nucleotide sequence of FIG. 54 (SEQ ID NO: 42) in the ORF. pT2C-0001.

    10) -1-2 Construction of HT1-11scFv / C3E-3007 bispecific molecule (T2C-0003) expression vector    

In the same manner as in Example 10) -1-1, an anti-TROP2-CD3 bispecific molecular expression vector containing the nucleotide sequence of Fig. 55 (SEQ ID NO: 44) in the ORF was constructed. However, pC3E-3007 was used as a template for making a vector fragment. The obtained expression vector was named "pT2C-0003".

    10) -1-3 Construction of HT1-11scFv / C3E-7035 bispecific molecule (T2C-0005) expression vector    

In the same manner as in Example 10) -1-1, an anti-TROP2-CD3 bispecific molecular expression vector containing the nucleotide sequence of FIG. 56 (SEQ ID NO: 46) in the ORF was constructed. However, pC3E-7035 was used as a template when creating a vector fragment. The obtained expression vector was named "pT2C-0005".

    10) -1-4 Construction of HT1-11scFv / C3E-7036 bispecific molecule (T2C-0006) expression vector    

In the same manner as in Example 10) -1-1, an anti-TROP2-CD3 bispecific molecular expression vector containing the nucleotide sequence of FIG. 57 (SEQ ID NO: 48) in the ORF was constructed. However, as a template for making a vector fragment, pC3E-7036 was used. The obtained expression vector was named "pT2C-0006".

    10) -2 Performance of anti-TROP2-CD3 bispecific molecule‧Purification    

The performance and purification of T2C-0001, T2C-0003, T2C-0005, and T2C-0006 were performed in the same manner as in Example 4) -1-2. The amino acid sequence of T2C-0001 is described in Figure 58 (SEQ ID NO: 43). The amino acid sequence of T2C-0003 is described in Figure 59 (SEQ ID NO: 45). The amino acid sequence of T2C-0005 is described in Figure 60 (SEQ ID NO: 47). The amino acid sequence of T2C-0006 is described in Figure 61 (SEQ ID NO: 49).

    (Example 11) Evaluation of in vitro activity of anti-TROP2-CD3 bispecific molecule         11) -1 Evaluation of binding activity using SPR         11) -1-1 Anti-TROP2-CD3 bispecific molecule binding to TROP2    

The binding of the bispecific molecule and TROP2 was measured using a BIAcore 3000 (GE Healthcare Bioscience), an antigen captured with an anti-human IgG antibody, and a capture method using the bispecific molecule as an analyte. The antigenic line used a recombinant human TROP-2 / human IgG F fusion (R & D system). An anti-human IgG (Fc) antibody (Human Antibody Capture Kit, GE Healthcare Bioscience) was covalently bonded to a sensor chip CM5 (GE Healthcare Bioscience) at about 2000 RU by an amine coupling method. It is similarly fixed to a reference unit. HBS-P (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) was used as the running buffer. Bispecific molecules were prepared from 200 nM to 1 nM at a 2-fold dilution. The anti-human IgG (Fc) antibody was immobilized on the wafer. After adding 1 μg / ml of the antigen in about 30 seconds, the bispecific molecules of each concentration were added at a flow rate of 30 μl / minute for 300 seconds, and the dissociation phase was monitored for 600 seconds . As a regeneration solution, a 3M magnesium chloride solution was added for 30 seconds. For the analysis of the data, a 1: 1 combination model of the analysis software (BIAevaluation software, version 4.1.1) was used to calculate the association velocity constant ka, dissociation velocity constant kd, and dissociation constant (KD; KD = kd / ka).

    11) -1-2 Anti-TROP2-CD3 bispecific molecule binding to human CD3εγ single chain antigen    

The binding system of the bispecific molecule and the CD3εγ antigen was measured using a BIAcore 3000 (GE Healthcare Bioscience) on the immobilized antigen using an antibody as an analyte. As the antigen, a human CD3εγ single-chain antigen prepared by 2) -2-1 was used, and about 100 RU was covalently bonded to the sensor chip CM5 (GE Healthcare Bioscience) using an amine coupling method. No antigen protein was added to the reference unit and only immobilization was performed. HBS-P (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) was used as the running buffer. Bispecific molecules are prepared by diluting from the highest concentration of 1 μM by a 2-fold dilution to 4 nM, or by 2-fold dilution from 200 nM to 1 nM. On the wafer on which the antigen was immobilized, bispecific molecules of each concentration were added at a flow rate of 10 μl / min for 25 minutes, and the amount of binding was detected. As a regeneration solution, a 10 mM glycine acid solution pH 1.5 was added for 30 seconds. For data analysis, the analysis constant (BIAevaluation software, version 4.1.1) was used to calculate the dissociation constant KD from the binding amount at each concentration. The results are shown in Tables 2 and 3.

    11) -2 Evaluation of binding activity by flow cytometry         11) -2-1 Anti-TROP2-CD3 bispecific molecule binding to TROP2    

Using the same cancer cell line as in Example 9, staining and analysis were performed in the same manner. As shown in FIG. 62, an anti-TROP2-CD3 bispecific molecule was shown to bind to TROP2.

    11) Binding of 2-2 anti-TROP2-CD3 bispecific molecule to human CD3εγ single chain antigen    

Dyeing and analysis were performed in the same manner as in Example 7) -1-1. As shown in Fig. 63, it is shown that the anti-TROP2-CD3 bispecific molecule binds to a human CD3εγ single chain antigen.

    11) Binding of anti-TROP2-CD3 bispecific molecule to CD3 antigen of cynomolgus monkeys    

The cynomolgus cynomolgus PBMC obtained in Example 7) -2-1 was adjusted to an appropriate concentration in PBS containing 5% FBS, and stained and analyzed in the same manner as in Example 7) -1-1. As shown in Fig. 64, anti-TROP2-CD3 bispecific molecules (T2C-0001, T2C-0005, T2C-0006) were shown to bind to the cynomolgus CD3 antigen.

    11) Evaluation of cytotoxic activity of anti-TROP2-CD3 bispecific molecule         11) Analysis of TROP2 expression in -3-1 target cells    

Pharyngeal flat epithelial cancer cell line FaDu (ATCC), pancreatic cancer cell line HPAF-II (ATCC), and human lung cancer cell line Calu-6 were adjusted to an appropriate concentration with 5% FBS in PBS, and LIVE / DEAD Fixable Dead Cell was added Stain Kit (Thermo / Death Fixable Dead Cell Dye Kit) (Thermo Fisher Scientific), left to stand at 4 ° C for 30 minutes. After washing twice with 5% FBS-containing PBS, it was adjusted to a concentration of 2 × 10 ⁶ cells / mL with 5% FBS-containing PBS, and inoculated at 100 μL / well to a 96-well U-bottom microplate, centrifuged, and removed. Serum. An anti-TROP2 Alexa Fluor 488 antibody (eBioscience) and an isotype control antibody (eBioscience) diluted with 5% FBS-containing PBS were added to 25 μL / well, and allowed to stand at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS, it was resuspended in 5% FBS-containing PBS and detected by a flow cytometer (Cytomics FC500, Beckman Coulter). Data analysis was performed with Flowjo (Treestar), and the geometric mean fluorescence intensity (geometric MFI) of Alexa Fluor 488 in the dead cell removal fraction was calculated. As shown in Fig. 65 (A, B, and C), the performance of TROP2 was observed in FaDu and HPAF-II; no expression was observed in Calu-6.

    11) Modulation of -3-2 target cells    

FaDu, HPAF-II, and Calu-6 were adjusted to a concentration of 2 × 10 ⁶ cells / mL with 10% RPMI1640 medium (Thermo Fisher Scientific), and 100 μL of Chromium-51 Radionuclide (PerkinElmer) was added to each cell suspension. Company), cultured at 37 ° C, 5% CO ₂ for 2 hours. After washing twice with RPMI1640 medium containing 10% FBS, RPMI1640 medium containing 10% FBS was resuspended to 2 × 10 ⁵ cells / mL, and the target cells were used.

    11) Modulation of 3--3 effector cells    

A commercially available frozen PBMC (Cellular Technology Limited) was thawed at 37 ° C, transferred to a solution containing 10% FBS in RPMI1640 medium, and a solution of Anti-aggregate Wash reagent (Cellular Technology Limited) was added. After washing twice, The RPMI1640 medium containing 10% FBS was prepared as 1 × 10 ⁶ cells / mL and used as an effector cell.

    11) -3-4 Cytotoxicity test    

FaDu, HPAF-II, and Calu-6 obtained in Example 11) -3-2 were added to a 96-well U-bottom microplate at 50 μL / well. Various anti-TROP2-CD3 bispecific molecules prepared at various concentrations were added at 50 μL / well to the effector cells prepared at 11) -3-1-3 at 100 μL / well, and centrifuged at room temperature at 1000 rpm for 1 minute. Incubate at 37 ° C and 5% CO ₂ for 20-24 hours. 50 μL of the supernatant was recovered in a solid-state scintillation quantitative plate (LumaPlate) (PerkinElmer), and dried at 50 ° C. for about 2 hours, and then measured with a flat plate reader (TopCount: PerkinElmer). The cell lysis rate was also calculated by the following formula.

Cell lysis rate (%) = (A-B) / (C-B) × 100

A: Counting of sample wells

B: Mean (n = 3) of background (non-antibody added well) counts. When the antibody was added, 50 μL of a test medium was added. Other than that, the same operation was performed as for the sample well.

C: Mean (n = 3) count of maximum release (wells in which target cells were lysed with surfactant). When the antibody was added, 50 μL of the test medium was added. 100 μL of a surfactant was added, and 50 μL was transferred to a solid-state scintillation quantitative disk in the same manner as a sample well, and the measurement was performed.

As shown in FIGS. 66 (A, B, and C), FaDu and its cytotoxic activity against various TROP2-CD3 bispecific molecules of HPAF-II are shown). On the other hand, no cytotoxic activity on Calu-6 was observed.

    (Example 12) Preparation of anti-Axl-CD3 bispecific molecule         12) Construction of anti-Axl-CD3 bispecific molecular expression vector         12) -1-1 Construction of 11D5-T3scFv / C3E-7034 bispecific molecule (AXC-0001) expression vector    

A glycine sequence is added to the N-terminus of h # 11D5-T3H (described in FIG. 12 of European Patent Application Publication No. 2270053), and h # 11D5-T3L (described in European Patent Application Publication No. 2270053 Figure 6) sequence, an anti-Axl single chain antibody linked via a polypeptide linker composed of (GGGGS) triple repeats, designed the amino acid sequence of 11D5-T3scFv, and synthesized a nucleotide sequence encoding this (GENEART , Thermo Fisher Science). Using this as a template, a part of the human antibody heavy chain message sequence was attached to the 5 ′ side, and a linker between the scFvs was attached to the 3 ′ side using a designed primer, and PCR was performed to obtain an inserted DNA fragment. In addition, the expression vector pC3E-7034 prepared in 4) -3-1 will be used as a template, and the primers prepared by the PCR method using the human antibody heavy chain message sequence and the nucleotide sequence encoding anti-CD3 scFv will contain anti-CD3 scFv primers. CD3 scFv was amplified in the entire region of the vector to obtain a vector DNA fragment. The respective DNA fragments were combined using an In-Fusion HD selection kit (CLONTECH) to produce an anti-Axl-CD3 bispecific molecule containing the nucleotide sequence shown in FIG. 97 (SEQ ID NO: 89) in an ORF. Expression vector pAXC-0001.

    12) -1-2 Construction of 11D5-T3scFv / C3E-7036 bispecific molecule (AXC-0002) expression vector    

In the same manner as in Example 10) -1-1, an anti-Axl-CD3 bispecific molecular expression vector containing the nucleotide sequence shown in FIG. 99 (SEQ ID NO: 91) contained in the ORF was constructed. However, when creating a vector fragment, pC3E-7036 was used as a template. The obtained expression vector was named "pAXC-0002".

    12) -2 Anti-Axl-CD3 bispecific molecule performance and purification    

The expression and purification of AXC-0001 and AXC-0002 were performed in the same manner as in Example 4) -1-2. The amino acid sequence of AXC-0001 is shown in Figure 98 (SEQ ID NO: 90). The amino acid sequence of AXC-0002 is shown in Figure 100 (SEQ ID NO: 92).

    (Example 13) Evaluation of in vitro activity of anti-Axl-CD3 bispecific molecules         13) Analysis of Axl expression in -1 target cells    

Human lung cancer cell line A549 (ATCC), human pancreatic cancer cell line PANC-1 (ATCC), MIA PaCa-2 (ATCC), human myeloma cell line U266B1 (ATCC), and mantle cell lymphoma cell line Jeko- 1 (ATCC) was adjusted to an appropriate concentration with 5% FBS-containing PBS, and a LIVE / DEAD Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) was added, and left to stand at 4 ° C for 30 minutes. After washing twice with 5% FBS-containing PBS, it was adjusted to a concentration of 2 × 10 ⁶ cells / mL with 5% FBS-containing PBS, and inoculated at 100 μL / well to a 96-well U-bottom microplate, centrifuged, and removed. Serum. Anti-Axl antibody (RD-Systems) and Isotype Control antibody (RD-Systems) diluted with 5% FBS-containing PBS were added at 25 μL / well, and allowed to stand at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS, Alexa Fluor488 anti-mouse IgG antibody (Thermo Fisher Scientific) diluted with 5% FBS-containing PBS was added at 25 μL / well, and left at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS, it was resuspended in 5% FBS-containing PBS, and detected by flow cytometry (Cytomics FC500, Beckman Coulter). Data analysis was performed with Flowjo (Treestar), and the geometric mean fluorescence intensity (geometric MFI) of Alexa Fluor 488 from which dead cells were removed was calculated. As shown in Fig. 107 (A, B, C, D, and E), the performance of Axl was observed on A549, PANC-1, and MIA PaCa-2, and no performance was observed on U266B1 and Jeko-1.

    13) Modulation of -2 target cells    

A549, PANC-1, MIA PaCa-2, U266B1, and Jeko-1 were prepared in RPMI1640 medium (Thermo Fisher Scientific) containing 10% FBS to a concentration of 2 × 10 ⁶ cells / mL, and the cell suspension was added every 1 mL 100 μL of Chromium-51 Radionuclide (PerkinElmer) was cultured at 37 ° C and 5% CO ₂ for 2 hours. After washing twice with RPMI1640 medium containing 10% FBS, the target cells were resuspended in RPMI1640 medium containing 10% FBS to 2 × 10 ⁵ cells / mL.

    13) Modulation of -3 effector cells    

A commercially available frozen PBMC (Cellular Technology Limited) was thawed at 37 ° C, transferred to a RPMI1640 medium containing 10% FBS, and the solution was added twice with an Anti-aggregate Wash reagent (Cellular Technology Limited), and then washed with RPMI1640 medium with 10% FBS was prepared as 1 × 10 ⁶ cells / mL as effector cells.

    13) -4 Cytotoxicity test    

A549, PANC-1, MIA PaCa-2, U266B1, and Jeko-1 obtained in Example 13) -2 were added to a 96-well U-bottom microplate at 50 μL / well. Various anti-Axl-CD3 bispecific molecules prepared at various concentrations were added at 50 μL / well, and effector cells modulated by 13) -3 were added at 100 μL / well. Centrifuge at room temperature for 1000 rpm × 1 minute. Incubate at 5% CO ₂ for 20-24 hours. 50 μL of the supernatant was recovered in a solid-state scintillation quantitative disk (PerkinElmer), and dried at 50 ° C. for about 2 hours, and then measured with a flat-plate reader (TopCount: PerkinElmer). The cell lysis rate was calculated by the following formula.

Cell lysis rate (%) = (A-B) / (C-B) × 100

A: Counting of sample wells.

B: Mean (n = 3) of background (non-antibody added well) counts. When the antibody was added, 50 μL of a test medium was added. Except for this, perform the same operation as the sample well.

C: Mean (n = 3) count of maximum release (wells in which target cells were lysed with surfactant). When antibody was added, 50 μL of test medium was added. 100 μL of a surfactant was added, and 50 μL was transferred to a solid-state scintillation quantitative disk in the same manner as a sample well, and the measurement was performed.

As shown in FIG. 108 (A, B, C, D, E), various anti-Axl-CD3 bispecific molecules exhibit cytotoxic activity against A549, PANC-1, and MIA PaCa-2. On the other hand, no cytotoxic activity was observed against U266B1 and Jeko-1.

    (Example 14) Preparation of anti-HLA-A2 / MAGEC1-CD3 bispecific molecular expression vector         14) -1 Construction of anti-HLA-A2 / MAGEC1-CD3 bispecific molecular expression vector         14) -1-1 Construction of MAG-032scFv / C3E-7034 bispecific molecule (MGC-0001) expression vector    

HLA-A2 / MAGEC1 specifically binds MAG-032 scFv from human antibody phage library. Using the nucleotide sequence encoding the amino acid sequence of MAG-032 scFv as a template, a PCR method using designed primers was used to attach a part of the human antibody heavy chain message sequence to the 5 'side and to connect the scFv between the 3' side. And a nucleotide sequence encoding a part of C3E-7034 to obtain an inserted DNA fragment containing a nucleotide sequence encoding an amino acid sequence of MAG-032 scFv. The expression vector pC3E-7034 prepared in 4) -3-1 will be used as a template, and a primer composed of a human antibody heavy chain message sequence and a nucleotide sequence encoding an amino terminal sequence of an anti-CD3 scFv will be used, and the PCR method will be used. The entire region of the vector containing the anti-CD3 scFv was amplified to obtain a vector DNA fragment. Anti-HLA-A2 / MAGEC1-CD3 including the nucleotide sequence shown in FIG. 101 (SEQ ID NO: 93) in the ORF was prepared by combining the respective DNA fragments using an In-Fusion HD selection kit (CLONTECH). The bispecific molecular expression vector pMGC-0001.

    14) -1-2 Construction of MAG-032scFv / C3E-7036 bispecific molecule (MGC-0002) expression vector    

In the same manner as in Example 14) -1-1, an anti-HLA-A2 / MAGEC1-CD3 bispecific molecular expression vector containing the nucleotide sequence shown in FIG. 103 (SEQ ID NO: 95) in an ORF was constructed. However, pC3E-7036 was used as a template for making the vector fragment. The obtained expression vector was named "pMGC-0002".

    14) -2 Performance of anti-HLA-A2 / MAGEC1-CD3 bispecific molecule‧Purification    

The expression and purification of MGC-0001 and MGC-0002 were performed in the same manner as in Example 4) -1-2. The amino acid sequence of MGC-0001 is shown in Figure 102 (SEQ ID NO: 94). The amino acid sequence of MGC-0002 is described in Figure 104 (SEQ ID NO: 96).

    (Example 15) Evaluation of in vitro activity of anti-HLA-A2 / MAGEC1-CD3 bispecific molecule         15) Performance analysis of HLA-A2 / MAGEC1 in -1 target cells    

Human lymphoblast fusion cell line T2 (ATCC) cells were adjusted to an appropriate concentration in AIM-V medium (Thermo Fisher Scientific) containing 20% FBS, and Figure 106 (SEQ ID NO: 97) MAGEC1 peptide (Sigma Genosys) was added. Or DMSO, incubate at 37 ° C for 4 hours. After washing twice with AIM-V medium containing 20% FBS, it was adjusted to the appropriate concentration with PBS containing 5% FBS, and a LIVE / DEAD Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) was added, and left to stand at 4 ° C for 30 minutes. minute. After washing twice with 5% FBS-containing PBS, the solution was adjusted to a concentration of 2 × 10 ⁶ cells / mL with 5% FBS-containing PBS, and 100 μL / well was inoculated into a 96-well U-bottom microplate, and centrifuged to remove it. Serum. An anti-HLA-A2 / MAGEC1 antibody (MAG032 scFv) diluted with 5% FBS-containing PBS was added at 25 μL / well, and allowed to stand at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS, it was diluted with 5% FBS-containing PBS. Penta-His Alexa Fluor 488 (QIAGEN) was added at 25 μL / well, and left at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS, it was resuspended in 5% FBS-containing PBS, and detected by flow cytometry (Cytomics FC500, Beckman Coulter). Data analysis was performed with Flowjo (Treestar), and the geometric mean fluorescence intensity (geometric MFI) of Alexa Fluor 488 from which dead cells were removed was calculated. As shown in Fig. 109 (A, B), the expression of HLA-A2 / MAGEC1 was observed in T2 cells to which MAGEC1 peptide was added, and no expression was observed in T2 cells to which DMSO was added.

    15) Modulation of -2 target cells    

T2 cells were adjusted to an appropriate concentration in AIM-V medium (Thermo Fisher Scientific) containing 20% FBS, MAGEC1 peptide or DMSO was added, and incubated at 37 ° C for 4 hours. RPMI1640 medium (Thermo Fisher Scientific) containing 10% FBS was prepared to a concentration of 2 × 10 ⁶ cells / mL. 100 μL of Chromium-51 Radionuclide (PerkinElmer) was added to the cell suspension at 37 ° C, 5% CO. ₂ cultured under conditions of 2 hours. After washing twice with RPMI1640 medium containing 10% FBS, the target cells were resuspended in RPMI1640 medium containing 10% FBS to 2 × 10 ⁵ cells / mL.

    15) -3 modulation of effector cells    

A commercially available frozen PBMC (Cellular Technology Limited) was thawed at 37 ° C, transferred to a solution containing 10% FBS in RPMI1640 medium and added with an Anti-aggregate Wash reagent (Cellular Technology Limited). After washing twice, RPMI1640 medium with 10% FBS was prepared as 1 × 10 ⁶ cells / mL as effector cells.

    15) -4 Cytotoxicity test    

T2 cells obtained in Example 15) -2 were added to a 96-well U-bottomed microtiter plate at 50 μL / well. 50 μL / well was added to each concentration of various anti-HLA-A2 / MAGEC1-CD3 bispecific molecules, and 100 μL / well was added to effector cells modulated by 15) -3. After centrifugation at 1000 rpm for 1 minute at room temperature Incubate at 37 ° C and 5% CO ₂ for 20-24 hours. 50 μL of the supernatant was recovered in a solid-state scintillation quantitative disk (PerkinElmer), and dried at 50 ° C. for about 2 hours, and then measured with a flat-plate reader (TopCount: PerkinElmer). The cell lysis rate was calculated by the following formula.

Cell lysis rate (%) = (A-B) / (C-B) × 100

A: Counting of sample wells.

B: Mean (n = 3) of background (non-antibody added well) counts. When the antibody was added, 50 μL of a test medium was added. Except for this, perform the same operation as the sample well.

C: Mean (n = 3) count of maximum release (wells in which target cells were lysed with surfactant). When the antibody was added, 50 μL of the test medium was added. 100 μL of a surfactant was added, and 50 μL was transferred to a solid-state scintillation quantitative disk in the same manner as a sample well, and the measurement was performed.

As shown in Fig. 110 (A, B), various cytotoxic activities of various anti-HLA-A2 / MAGEC1-CD3 bispecific molecules on T2 cells to which MAGEC1 peptide is added are shown. On the other hand, no cytotoxic activity was observed on DMSO-added T2 cells.

Industrial availability

The humanized anti-CD3 antibody of the present invention is suitable for non-clinical trials for the development of medicine because it binds to human CD3 and also binds to cynomolgus CD3.

    [Non-keyword text of sequence list]    

SEQ ID NO: 1: Amino acid sequence of human CD3ε

SEQ ID No. 2: Amino acid sequence of human CD3δ

SEQ ID NO: 3 Amino acid sequence of human CD3γ

SEQ ID NO: 4: Nucleotide sequence encoding human CD3εγ single chain antigen

SEQ ID NO: 5: Amino acid sequence of His-scCD3 antigen

SEQ ID NO: 6: Nucleotide sequence encoding the heavy chain variable region of C3-147

SEQ ID NO: 7 (C3-147_VH AA): Amino acid sequence of C3-147 heavy chain variable region

SEQ ID NO: 8 (C3-147_VL DNA): Nucleotide sequence encoding the variable region of the light chain of C3-147

SEQ ID NO: 9 (C3-147_VL AA): Amino acid sequence of light chain variable region of C3-147

Sequence ID 10: (G4S linker sense):

Sequence identification number 11: (G4S linker antisence):

SEQ ID NO: 12 (C3E-7000_VH AA): Amino acid sequence of the heavy chain variable region of C3E-7000

SEQ ID NO: 13 (C3E-7000_VL AA): Amino acid sequence of the light chain variable region of C3E-7000

SEQ ID NO: 14 (C3E-7000 ORF): Nucleotide sequence encoding C3E-7000

SEQ ID NO: 15 (C3E-7000 AA): Amino acid sequence of C3E-7000

SEQ ID NO: 16: (C3E-7034_VH AA): Amino acid sequence of the heavy chain variable region of C3E-7034

SEQ ID NO: 17 Amino acid sequence of light chain variable region of C3E-7034

SEQ ID NO: 18 (C3E-7034 ORF): Nucleotide sequence encoding C3E-7034

SEQ ID NO: 19 (C3E_7034 AA): Amino acid sequence of C3E-7034

SEQ ID NO: 20: (C3E-7035_VL AA): Amino acid sequence of the light chain variable region of C3E-7035

SEQ ID NO: 21 (C3E-7035 ORF): Nucleotide sequence encoding C3E-7035

SEQ ID NO: 22: (C3E_7035 AA): Amino acid sequence of C3E-7035

SEQ ID NO: 23 (C3E-7036_VL_AA): Amino acid sequence of the light chain variable region of C3E-7036

SEQ ID NO: 24 (C3E-7036 ORF): Nucleotide sequence encoding C3E-7036

SEQ ID NO: 25: (C3E_7036 AA): Amino acid sequence of C3E-7036

SEQ ID NO: 26 (7000_CDR-H1): Amino acid sequence of CDR-H1 of C3E-7000 series

SEQ ID NO: 27 (7000_CDR-H2): Amino acid sequence of CDR-H2 of C3E-7000 series

SEQ ID NO: 28: (7000_CDR-H3): Amino acid sequence of CDR-H3 of C3E-7000 series

SEQ ID NO: 29 (7000_CDR-L1): Amino acid sequence of CDR-L1 of C3E-7000 series

SEQ ID NO: 30: (7000_CDR-L2): Amino acid sequence of CDR-L2 of C3E-7000 series

SEQ ID NO: 31: (7000_CDR-L3): Amino acid sequence of CDR-L3 of C3E-7000 series

SEQ ID NO: 32 (C3E-3000 ORF): Nucleotide sequence encoding OKT3 scFv

SEQ ID NO: 33 (C3E-3000 AA): Amino acid sequence of OKT3 scFv

SEQ ID NO: 34 (C3E-3007 ORF): Nucleotide sequence encoding C3E-3007 scFv

SEQ ID NO: 35: (C3E-3007 AA): Amino acid sequence of C3E-3007 scFv

SEQ ID NO: 36: (C3E-3000_VH AA): Amino acid sequence of the variable region of the heavy chain of OKT3

SEQ ID NO: 37: (C3E-3000_VL AA): Amino acid sequence of the light chain variable region of OKT3

SEQ ID NO: 38: (C3E-3007_VH AA): Amino acid sequence of the heavy chain variable region of C3E-3007

SEQ ID NO: 39 (C3E-3007_VL AA): Amino acid sequence of the light chain variable region of C3E-3007

SEQ ID NO: 40 (HT1-11 ORF): Nucleotide sequence encoding HT1-11 scFv

SEQ ID NO: 41: (HT1-11 AA): Amino acid sequence of HT1-11 scFv

SEQ ID NO: 42 (T2C-0001 ORF): ORF nucleotide sequence encoding T2C-0001

SEQ ID NO: 43 (T2C-0001 AA): Amino acid sequence of T2C-0001

SEQ ID NO: 44 (T2C-0003 ORF): ORF nucleotide sequence encoding T2C-0003

SEQ ID NO: 45 (T2C-0003 AA): Amino acid sequence of T2C-0003

SEQ ID NO: 46 (T2C-0005 ORF): ORF nucleotide sequence encoding T2C-0005

SEQ ID NO: 47: (T2C-0005 AA): Amino acid sequence of T2C-0005

SEQ ID NO: 48: (T2C-0006 ORF): ORF nucleotide sequence encoding T2C-0006

SEQ ID NO: 49 (T2C-0006 AA): Amino acid sequence of T2C-0006

SEQ ID NO: 50 Amino acid sequence of sense primer for heavy chain gene amplification

SEQ ID NO: 51 Nucleotide sequence of the first antisense primer for heavy chain gene amplification

SEQ ID NO: 52 Nucleotide sequence of the second antisense primer for heavy chain gene amplification

SEQ ID No. 53: Nucleotide sequence of a sense primer for light chain gene amplification

SEQ ID NO: 54 Nucleotide sequence of the first antisense primer for light chain gene amplification

SEQ ID NO: 55: Nucleotide sequence of the second antisense primer for light chain gene amplification

SEQ ID No. 56: Nucleotide sequence of a sense primer for heavy chain sequencing

SEQ ID NO: 57: Nucleotide sequence of antisense primer 1 for light chain sequencing

SEQ ID NO: 58 Nucleotide sequence of antisense primer 2 for light chain sequencing

SEQ ID NO: 59: ORF nucleotide sequence encoding C3E-7078

SEQ ID NO: 60: Amino acid sequence of C3E-7078

SEQ ID NO: 61: ORF nucleotide sequence encoding C3E-7079

SEQ ID NO: 62: Amino acid sequence of C3E-7079

SEQ ID NO: 63: ORF nucleotide sequence encoding C3E-7085

SEQ ID NO: 64: Amino acid sequence of C3E-7085

SEQ ID NO: 65: ORF nucleotide sequence encoding C3E-7086

SEQ ID NO: 66: Amino acid sequence of C3E-7086

SEQ ID NO: 67: ORF nucleotide sequence encoding C3E-7087

SEQ ID NO: 68: Amino acid sequence of C3E-7087

SEQ ID NO: 69: ORF nucleotide sequence encoding C3E-7088

SEQ ID NO: 70: Amino acid sequence of C3E-7088

SEQ ID NO: 71: ORF nucleotide sequence encoding C3E-7089

SEQ ID NO: 72: Amino acid sequence of C3E-7089

SEQ ID NO: 73: ORF nucleotide sequence encoding C3E-7090

SEQ ID NO: 74: Amino acid sequence of C3E-7090

SEQ ID NO: 75: ORF nucleotide sequence encoding C3E-7091

SEQ ID NO: 76: Amino acid sequence of C3E-7091

SEQ ID NO: 77: ORF nucleotide sequence encoding C3E-7092

SEQ ID NO: 78: Amino acid sequence of C3E-7092

SEQ ID No. 79: ORF nucleotide sequence encoding C3E-7093

SEQ ID NO: 80: Amino acid sequence of C3E-7093

SEQ ID NO: 81: ORF nucleotide sequence encoding C3E-7094

SEQ ID NO: 82: Amino acid sequence of C3E-7094

SEQ ID NO: 83: ORF nucleotide sequence encoding C3E-7095

SEQ ID NO: 84: Amino acid sequence of C3E-7095

SEQ ID NO: 85: Nucleotide sequence of HN53R Fw

SEQ ID NO: 86: Nucleotide sequence of HN53R Rv

SEQ ID NO: 87: Nucleotide sequence of HN53S Fw

SEQ ID NO: 88: The nucleotide sequence of HN53S Rv

SEQ ID NO: 89 (AXC-0001 ORF): ORF nucleotide sequence encoding AXC-0001

SEQ ID NO: 90: (AXC-0001 AA): Amino acid sequence of AXC-0001

SEQ ID NO: 91 (AXC-0002 ORF): ORF nucleotide sequence encoding AXC-0002

SEQ ID NO: 92 (AXC-0002 AA): Amino acid sequence of AXC-0002

SEQ ID NO: 93 (MGC-0001 ORF): ORF nucleotide sequence encoding MGC-0001

SEQ ID NO: 94 (MGC-0001 AA): Amino acid sequence of MGC-0001

SEQ ID NO: 95 (MGC-0002 ORF): ORF nucleotide sequence encoding MGC-0002

SEQ ID NO: 96 (MGC-0002 AA): Amino acid sequence of MGC-0002

SEQ ID NO: 97 (MAGEC1 peptide): Amino acid sequence of MAGEC1 peptide

SEQ ID No. 98: (CDRH2 of the variant): amino acid sequence of the CDR variant CDRH2

SEQ ID NO: 99 (CDRL2 of the variant): Amino acid sequence of the CDR variant CDRL2

SEQ ID NO: 100: (VH of C3E-7034 variant): Amino acid sequence of heavy chain variable region of CDR variant of C3E-7034

SEQ ID NO: 101 (VL of C3E-7034 variant): Amino acid sequence of light chain variable region of CDR variant of C3E-7034

SEQ ID NO: 102: (VL of C3E-7035 variant): Amino acid sequence of light chain variable region of CDR variant of C3E-7035

SEQ ID NO: 103 (VL of C3E-7036 variant): Amino acid sequence of light chain variable region of CDR variant of C3E-7036

<110> Daiichi Sankyo Co., Ltd.

<120> Anti-CD3 antibody and molecule containing the same

<130> FP1729

<150> JP2016-249148

<151> 2016-12-22

<160> 103

<170> PatentIn version 3.5

<210> 1

<211> 207

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 171

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3

<211> 182

<212> PRT

<213> Homo sapiens

<400> 3

<210> 4

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> CD3 ε γ DNA

<400> 4

<210> 5

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> His-scCD3 AA

<400> 5

<210> 6

<211> 354

<212> DNA

<213> Rutilus rutilus

<400> 6

<210> 7

<211> 118

<212> PRT

<213> Rutilus rutilus

<400> 7

<210> 8

<211> 327

<212> DNA

<213> Rutilus rutilus

<400> 8

<210> 9

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> C3-147_VL AA

<400> 9

<210> 10

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> G4S Linker Meaning

<400> 10

<210> 11

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> G4sS Linker Antonym

<400> 11

<210> 12

<211> 118

<212> PRT

<213> Rutilus rutilus

<400> 12

<210> 13

<211> 109

<212> PRT

<213> Rutilus rutilus

<400> 13

<210> 14

<211> 867

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7000 ORF

<400> 14

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<211> 270

<212> PRT

<213> Artificial sequence

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<223> C3E-7000 AA

<400> 15

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<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7034_VH AA

<400> 16

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<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7034_VL AA

<400> 17

<210> 18

<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7034 ORF

<400> 18

<210> 19

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7034 AA

<400> 19

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<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7035_VL AA

<400> 20

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<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7035 ORF

<400> 21

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<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7035 AA

<400> 22

<210> 23

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7036_VL AA

<400> 23

<210> 24

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<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7036 ORF

<400> 24

<210> 25

<211> 267

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7036 AA

<400> 25

<210> 26

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> 7000_CDR-H1

<400> 26

<210> 27

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> 7000_CDR-H2

<400> 27

<210> 28

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> 7000_CDR-H3

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<212> PRT

<213> Artificial sequence

<220>

<223> 7000_CDR-L1

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<213> Artificial sequence

<220>

<223> 7000_CDR-L2

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<223> 7000_CDR-L3

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<213> Artificial sequence

<220>

<223> C3E-3000 ORF

<400> 32

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<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-3000 AA

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<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-3007 ORF

<400> 34

<210> 35

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-3007 AA

<400> 35

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<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-3000_VH AA

<400> 36

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<212> PRT

<213> Artificial sequence

<220>

<223> C3E-3000_VL AA

<400> 37

<210> 38

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-3007_VH AA

<400> 38

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<212> PRT

<213> Artificial sequence

<220>

<223> C3E-3007_VL AA

<400> 39

<210> 40

<211> 822

<212> DNA

<213> Artificial sequence

<220>

<223> HT1-11 scFv ORF

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<220>

<223> HT1-11 scFv AA

<400> 41

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<212> DNA

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<220>

<223> T2C-0001 ORF

<400> 42

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<212> PRT

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<220>

<223> T2C-0001 AA

<400> 43

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<220>

<223> T2C-0003 ORF

<400> 44

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<220>

<223> T2C-0003 AA

<400> 45

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<220>

<223> T2C-0005 ORF

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<220>

<223> T2C-0005 AA

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<212> DNA

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<220>

<223> T2C-0006 ORF

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<210> 49

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<212> PRT

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<220>

<223> T2C-0006 AA

<400> 49

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<212> DNA

<213> Artificial sequence

<220>

<223> Nhe-polyC-S

<220>

<221> Various features that cannot be classified

<222> (37) .. (37)

<223> n is a, c, g or t

<400> 50

<210> 51

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> rIg γ -AS1

<400> 51

<210> 52

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<220>

<223> rIg γ -AS2

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<212> DNA

<213> Artificial sequence

<220>

<223> Nhe-polyC-S2

<220>

<221> Various features that cannot be classified

<222> (37) .. (37)

<223> n is a, c, g or t

<400> 53

<210> 54

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<212> DNA

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<220>

<223> rIgL-AS1

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<220>

<223> rIgL-AS2

<400> 55

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<212> DNA

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<220>

<223> rIg γ -seq

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<220>

<223> rIgL-seq1

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<220>

<223> rIgL-seq2

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<220>

<223> C3E-7078 ORF

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<220>

<223> C3E-7085 ORF

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<223> C3E-7085 AA

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<220>

<223> C3E-7086 ORF

<400> 65

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<220>

<223> C3E-7086 AA

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<220>

<223> C3E-7087 AA

<400> 68

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<220>

<223> C3E-7088 ORF

<400> 69

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<220>

<223> C3E-7088 AA

<400> 70

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<220>

<223> C3E-7089 ORF

<400> 71

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<220>

<223> C3E-7089 AA

<400> 72

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<220>

<223> C3E-7090 ORF

<400> 73

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<211> 269

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<220>

<223> C3E-7090 AA

<400> 74

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<213> Artificial sequence

<220>

<223> C3E-7091 ORF

<400> 75

<210> 76

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7091 AA

<400> 76

<210> 77

<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7092 ORF

<400> 77

<210> 78

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7092 AA

<400> 78

<210> 79

<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7093 ORF

<400> 79

<210> 80

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7093 AA

<400> 80

<210> 81

<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7094 ORF

<400> 81

<210> 82

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7094 AA

<400> 82

<210> 83

<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> C3E-7095 ORF

<400> 83

<210> 84

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> C3E-7095 AA

<400> 84

<210> 85

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> HN53R Fw

<400> 85

<210> 86

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> HN53R Rv

<400> 86

<210> 87

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> HN53S Fw

<400> 87

<210> 88

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> HN53S Rv

<400> 88

<210> 89

<211> 1590

<212> DNA

<213> Artificial sequence

<220>

<223> AXC-0001 ORF

<400> 89

<210> 90

<211> 511

<212> PRT

<213> Artificial sequence

<220>

<223> AXC-0001 AA

<400> 90

<210> 91

<211> 1584

<212> DNA

<213> Artificial sequence

<220>

<223> AXC-0002 ORF

<400> 91

<210> 92

<211> 509

<212> PRT

<213> Artificial sequence

<220>

<223> AXC-0002 AA

<400> 92

<210> 93

<211> 1620

<212> DNA

<213> Artificial sequence

<220>

<223> MGC-0001 ORF

<400> 93

<210> 94

<211> 521

<212> PRT

<213> Artificial sequence

<220>

<223> MGC-0001 AA

<400> 94

<210> 95

<211> 1614

<212> DNA

<213> Artificial sequence

<220>

<223> MGC-0002 ORF

<400> 95

<210> 96

<211> 519

<212> PRT

<213> Artificial sequence

<220>

<223> MGC-0002 AA

<400> 96

<210> 97

<211> 10

<212> PRT

<213> Homo sapiens

<400> 97

<210> 98

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> CDRH2 of the variant

<220>

<221> Xaa

<222> (3) .. (3)

<223> The first Xaa is any naturally occurring amino acid

<220>

<221> Xaa

<222> (4) .. (4)

<223> The second Xaa is any naturally occurring amino acid

<400> 98

<210> 99

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> CDRL2 of the variant

<220>

<221> X

<222> (2) .. (2)

<223> Xaa is any naturally occurring amino acid

<400> 99

<210> 100

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> VH of C3E-7034 variant

<220>

<221> Various features that cannot be classified

<222> (53) .. (53)

<223> The first Xaa is any naturally occurring amino acid

<220>

<221> Various features that cannot be classified

<222> (54) .. (54)

<223> The second Xaa is any naturally occurring amino acid

<400> 100

<210> 101

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> VL of C3E-7034 variant

<220>

<221> Various features that cannot be classified

<222> (52) .. (52)

<223> Xaa is any naturally occurring amino acid

<400> 101

<210> 102

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of C3E-7035 variant

<220>

<221> Various features that cannot be classified

<222> (52) .. (52)

<223> Xaa is any naturally occurring amino acid

<400> 102

<210> 103

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of C3E-7036 variant

<220>

<221> Various features that cannot be classified

<222> (52) .. (52)

<223> Xaa is any naturally occurring amino acid

<400> 103

Claims (55)

    An antibody or an antigen-binding fragment of the antibody, characterized in that the heavy chain sequence includes: a CDRH1 comprising an amino acid sequence represented by sequence identification number 26; a CDRH2 comprising an amino acid sequence represented by sequence identification number 98; and CDRH3 containing the amino acid sequence shown in SEQ ID NO: 28; light chain sequences each include: CDRL1 including the amino acid sequence shown in SEQ ID 29, and CDRL2 containing the amino acid sequence shown in SEQ ID 99 And CDRL3 containing the amino acid sequence shown in SEQ ID NO: 31; and binds to human CD3 and cynomolgus CD3.     The antibody or antigen-binding fragment of claim 1, wherein the first X _aa of the aforementioned CDRH2 is selected from the group consisting of (A, E, G, H, I, L, T, V, R, S) And the second X _aa is S, or the first X _aa is N, and the second X _aa is selected from (E, R, F, Y, L, V, I, K, T) In the group formed, the X _aa of the aforementioned CDRL2 is selected from the group consisting of (Q, A, G, S, N, D); and binds to human CD3 and cynomolgus monkey CD3. For example, the antibody or antigen-binding fragment of claim 1 or 2, wherein the first X _aa of the aforementioned CDRH2 is selected from the group consisting of (R, S), the second X _aa is S, and the foregoing X _aa of CDRL2 is selected from the group consisting of (Q, A, G, S, N, D); and binds to human CD3 and cynomolgus monkey CD3.     For example, the antibody or antigen-binding fragment of claim 1, wherein the heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, and the aforementioned CDRH1 is composed of an amino acid sequence shown in SEQ ID NO: 26, The CDRH2 is composed of an amino acid sequence shown in SEQ ID NO: 27, and the CDRH3 is composed of an amino acid sequence shown in SEQ ID NO: 28; and the light chain sequence includes a variable having CDRL1, CDRL2, and CDRL3. Region, the above-mentioned CDRL1 is composed of the amino acid sequence shown in SEQ ID NO: 29, the above-mentioned CDRL2 is composed of the amino acid sequence shown in SEQ ID NO: 30, and the above-mentioned CDRL3 is made of amine represented by SEQ ID NO: 31 Consisting of amino acid sequences; and binding to human CD3 and cynomolgus CD3.         The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the heavy chain variable region sequence comprises an amino acid sequence represented by sequence identification number 100.     The antibody or antigen-binding fragment thereof according to claim 5, wherein the first X _aa of the amino acid sequence shown by sequence identification number 100 is selected from (A, E, G, H, I, L, T, V , R, S), and the second X _aa is S, or the first X _aa is N, and the second X _aa is selected from (E, R, F, Y, L, V , I, K, T). The antibody or antigen-binding fragment thereof according to claim 5, wherein the first X _aa of the amino acid sequence represented by the sequence identification number 100 is selected from the group consisting of (R, S), and the second X _aa is S.     The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the light chain variable region comprises an amino acid sequence represented by any one of sequence identification numbers 101, 102, and 103.     The antibody or antigen-binding fragment thereof according to claim 8, wherein X _aa of the amino acid sequence shown by any one of sequence identification numbers 101, 102, and 103 is selected from (Q, A, G, S, N , D).     The antibody or the antigen-binding fragment thereof according to claim 5, wherein the heavy chain variable region sequence comprises an amino acid sequence represented by SEQ ID NO: 16.         The antibody or the antigen-binding fragment thereof according to claim 8, wherein the light chain variable region sequence comprises an amino acid sequence represented by any one of sequence identification numbers 17, 20, and 23.     The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: a heavy chain variable region comprising the amino acid sequence shown in sequence identification number 100; and any one of sequence identification numbers 101, 102, and 103 The light chain variable region of the amino acid sequence shown by one; the first X _aa of the amino acid sequence shown by sequence identification number 100 is selected from (A, E, G, H, I, L, T , V, R, S), and the second X _aa is S, or the first X _aa is N, and the second X _aa is selected from (E, R, F, Y, L , V, I, K, T), X _aa of the amino acid sequence shown by any one of the sequence identification numbers 101, 102, and 103 is selected from (Q, A, G, S, N, D). For example, the antibody or antigen-binding fragment thereof according to claim 12, wherein the first X _aa of sequence identification number 100 is selected from the group consisting of (R, S), and the second X _aa is S; and the sequence identification number X _aa of the amino acid sequence shown in any one of 101, 102, and 103 is selected from the group consisting of (Q, A, G, S, N, D).     If the antibody or antigen-binding fragment of claim 13 is: a heavy chain variable region comprising an amino acid residue comprising sequence identification number 60 2 to 119, and a sequence comprising 135 to An antibody to the light chain variable region of an amino acid residue of 243 or an antigen-binding fragment of the antibody; a heavy chain variable region containing an amino acid residue of SEQ ID NO: 64 to 119, and a sequence comprising the same Antibodies of the light chain variable regions of amino acid residues of IDs 64 to 135 to 241 or antigen-binding fragments of the antibodies; Antibodies or antigen-binding fragments of the variable region and light chain variable region of amino acid residues of amino acid residues 135 to 243 of sequence identification number 66; amino acids containing amino acid sequences of 2 to 119 of sequence identification number 68 The heavy chain variable region of the residue, and the antibody or the antigen-binding fragment thereof comprising the light chain variable region of the amino acid residues of amino acid residues of sequence numbers 68 to 135 to 243; Heavy chain variable region of amino acid residues from 119 to 119, and amino acids containing 135 to 243 of sequence identification number 70 Antibodies to light chain variable regions of residues or antigen-binding fragments of the antibodies; heavy chain variable regions containing amino acid residues containing sequence identification numbers 72 to 119, and 135 containing sequence identification numbers 72 to 135 An antibody to the light chain variable region of an amino acid residue to 243 or an antigen-binding fragment of the antibody; a heavy chain variable region comprising an amino acid residue comprising sequence identification number 74 2 to 119, and Antibodies to light chain variable regions of amino acid residues of SEQ ID NOs: 74 to 135 to 243 or antigen-binding fragments of the antibodies; heavy chains containing amino acid residues of SEQ ID Nos. 76 to 119 Antibodies or antigen-binding fragments of the variable region and the light chain variable region of amino acid residues comprising sequence identification numbers 76 to 135 to 243; the amino group comprising sequence identification numbers 78 to 119 An acid residue having a heavy chain variable region, and an antibody comprising the light chain variable region of amino acid residues of sequence identification numbers 78 to 243, or an antibody-binding fragment thereof; Heavy chain variable regions of amino acid residues from 2 to 119, and amine groups containing sequence identification numbers 135 to 243 Antibodies to light chain variable regions of residues or antigen-binding fragments of the antibodies; heavy chain variable regions containing amino acid residues containing sequence identification numbers 21-2 to 119, and sequence numbers 82 to 135 An antibody to a light chain variable region of an amino acid residue to 243 or an antigen-binding fragment of the antibody; or a heavy chain variable region containing an amino acid residue comprising sequence identification number 84 2 to 119, and An antibody or an antigen-binding fragment thereof comprising a light chain variable region of an amino acid residue of SEQ ID NO: 84 to 135 to 243.         For example, the antibody of claim 1, 4, 5, 8, 10, or 11 or the antigen-binding fragment of the antibody contains a heavy chain variable region including a amino acid sequence shown in SEQ ID NO: 16 and a linker ( linker), and a light chain variable region comprising an amino acid sequence shown in any one of sequence identification numbers 17, 20, and 23.         The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the variable region is bound from the amine terminal side of the antibody in the order of a heavy chain variable region, a light chain variable region, or lightly The chain variable region and the heavy chain variable region are sequentially combined, and arbitrarily: i) has a linker between the two variable regions; ii) the amino group terminal of the variable region on the amino terminal side has glycine Residues; iii) a linker, a FLAG tag, and / or a His tag are bonded to the carboxy terminus of the variable region on the carboxy terminus side.         For example, the antigen of claim 16 or the antigen-binding fragment of the antibody contains: an amino acid sequence including an amino acid residue at positions 2 to 243 of sequence identification number 60; Amino acid sequence of amino acid residue at position 241, amino acid sequence including amino acid residues at positions 2 to 243 of sequence identification number 66, amine including amino acid positions 2 to 243 of sequence identification number 68 Amino acid residues containing amino acid residues, amino acid residues containing amino acid residues at positions 2 to 243 of sequence identification number 70, amino acid residues including amino acid residues at positions 2 to 243 of sequence identification number 72 Amino acid sequence containing amino acid residues at positions 2 to 243 of sequence identification number 74, amino acid sequence containing amino acid residues at positions 2 to 243 of sequence identification number 76 Sequence, an amino acid sequence containing an amino acid residue at positions 2 to 243 of sequence identification number 78, an amino acid sequence containing an amino acid residue at positions 2 to 243 of sequence identification number 80, an included sequence Amino acid sequences of amino acid residues at positions 2 to 243 of ID 82, or amines containing amino acid residues of positions 2 to 243 of sequence identification 84 Acid sequence.         If the antigen of claim 16 or the antigen-binding fragment of the antibody contains: an amino acid sequence including amino acid residues of sequence identification number 2 to 269, and an amine including sequence identification number 2 to 269 Amino acid residues containing amino acid residues, amino acid residues containing amino acid residues 2 to 267 of sequence identification number 25, amino acid residues including amino acid residues of sequence identification number 2 to 269 Sequence, amino acid sequence containing amino acid residues 2 to 267 of sequence identification number 64, amino acid sequence containing amino acid residues of sequence identification number 2 to 269, amino acid sequence including sequence identification number 68 Amino acid sequences of amino acid residues 2 to 269, amino acid sequences including amino acid residues 2 to 269 of sequence identification number 70, amino acid residues including 2 to 269 of sequence identification number 72 Amino acid sequence including amino acid residues of 2 to 269 of sequence identification number 74, amino acid sequence including amino acid residues of 2 to 269 of sequence identification number 76, Amino acid sequences of amino acid residues of SEQ ID NO: 78 to 2 to 269, amino groups of amino acid residues containing SEQ ID NO: 80 to 2 of 269 Sequence comprising the amino acid sequence of SEQ ID NO amino acids 2-269 of 82 residues, or the amino acid sequence comprising SEQ ID NO 84 the amino acid residues of 2-269.         An antibody or an antigen-binding fragment of the antibody, comprising an amino acid sequence and binding to human CD3 and cynomolgus monkey CD3, wherein the amino acid sequence is encoded by a nucleotide sequence, the nucleotide sequence Polynucleotides that are hybridized under stringent conditions to the complementary strands of the following polynucleotides: encoding the amino acid sequence contained in the antibody of any one of claims 14 to 18 or the antigen-binding fragment of the antibody A polynucleotide of a nucleotide sequence.         An antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain contained in the antibody according to any one of claims 14 to 18 or the antigen-binding fragment of the antibody The amino acid sequence is 90% or more of the same amino acid sequence; and the light chain contains an amino group of the light chain contained in the antibody according to any one of claims 14 to 18 or an antigen-binding fragment of the antibody The acid sequence is more than 70% identical amino acid sequence; and it binds to human CD3 and cynomolgus monkey CD3.         An antibody or an antigen-binding fragment of the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises: an antibody-containing fragment comprising the antibody according to any one of claims 14 to 18 or an antigen-binding fragment of the antibody In the amino acid sequence of the heavy chain, one to several amino acids are substituted, deleted or added; and the light chain comprises: the antibody comprising any one of claims 14 to 18 Or the amino acid sequence of the light chain contained in the antigen-binding fragment of the antibody, one to several amino acids are substituted, deleted or added; and the same as human CD3 and cynomolgus CD3 Combined.         An antibody or an antigen-binding fragment of the antibody, which binds to the same site on human CD3 as the antibody of any of claims 14 to 18 or the antigen-binding fragment of the antibody, and binds to the cynomolgus monkey CD3 Combined.         An antibody or an antigen-binding fragment of the antibody that competes with the antibody according to any one of claims 14 to 18 or the antigen-binding fragment of the antibody for binding to human CD3 and binds to cynomolgus monkey CD3.         For example, the antibody or the antigen-binding fragment of claim 22, wherein the site on human CD3 to which the aforementioned antibody or the antigen-binding fragment of the antibody binds is the first in the amino acid sequence shown by sequence identification number 1. Serine at position 55, Glu at position 56, Glu at position 58 (Leu), tryptophan at position 59 (Trp), asparagus at position 65 Amino acid (Asn), isoleucine (Ile) at position 66, serine (Ser) at position 77, aspartic acid (Asp) at position 78, and arginine (Arg at position 101) ), Glycine (Gly) at position 101, serine (Ser) at position 103, lysine (Lys) at position 104, and proline (Pro) at position 105 The amino acid is composed of the above.         The antibody or antigen-binding fragment of any one of claims 1 to 17, 19 to 24, which is IgG.         The antibody or antigen-binding fragment of any one of claims 1 to 23, which is selected from the group consisting of Fab, F (ab) ', Fv, scFv, and sdAb.         The antibody or the antigen-binding fragment of any one of claims 1 to 17, 19 to 25, which is a humanized antibody or a human antibody comprising a human immunoglobulin constant region.         A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence of an antibody according to any one of claims 1 to 27 or an antigen-binding fragment of the antibody.         The polynucleotide of claim 27, which comprises a nucleotide sequence encoding the following amino acid sequence: an amino acid sequence comprising an amino acid residue at positions 2 to 243 of sequence identification number 19, and a sequence identification number Amino acid sequence of amino acid residues at positions 2 to 243 of 22, amino acid sequence including amino acid residues at positions 2 to 241 of sequence identification number 25, second sequence including sequence identification number 60 Amino acid sequences of amino acid residues from positions 243 to 243, amino acid sequences including amino acid residues of positions 2 to 241 of sequence identification number 64, and amino acid sequences of positions 2 to 243 of sequence identification number 66 Amino acid residues of amino acid residues, amino acid residues including amino acid residues at positions 2 to 243 of sequence identification number 68, amino acid residues including amino acid residues of positions 2 to 243 of sequence identification number 70 Amino acid sequence of amino group, amino acid sequence including amino acid residues at positions 2 to 243 of sequence identification number 72, amino acid sequence including amino acid residues of positions 2 to 243 of sequence identification number 74 Acid sequence, amino acid sequence containing amino acid residues 2 to 243 of sequence identification number 76, amino acid sequence containing amino acid positions 2 to 243 of sequence identification number 78 Amino acid sequence including amino acid residues at positions 2 to 243 of sequence identification number 80, amino acid sequence including amino acid residues at positions 2 to 243 of sequence identification number 82 An acid sequence, or an amino acid sequence comprising an amino acid residue at positions 2 to 243 of sequence identification number 84.         A vector comprising a polynucleotide according to any one of claims 28 or 29.         A cell comprising a polynucleotide according to any one of claims 28 or 29 or a vector according to claim 30, or producing an antibody according to any one of claims 1 to 27 or an antigen-binding fragment of the antibody.         A method for producing an antibody binding to human CD3 and cynomolgus CD3 or an antigen-binding fragment of the antibody, comprising the steps of culturing a cell as claimed in item 31; and recovering the antibody binding to human CD3 from the culture or A step of an antigen-binding fragment of the antibody.         An antibody that binds to human CD3 and cynomolgus CD3, or an antigen-binding fragment of the antibody, which is obtained by the method of claim 32.         A pharmaceutical composition comprising the antibody according to any one of claims 1 to 27 and 33 or an antigen-binding fragment of the antibody as an active ingredient.         An antigen-binding molecule comprising the antibody according to any one of claims 1 to 27, 33 or an antigen-binding fragment of the antibody.         As a numerator of claim 35, it is multispecific.         The molecule of claim 35 or 36, which comprises the antibody of any one of claims 1 to 27, 33 or an antigen-binding fragment of the antibody, and one or two or more additional antibodies or the antigen of the antibody Binding fragment.         The molecule of claim 37, wherein the antigen-binding fragment of the additional antibody is Fab, F (ab) ', Fv, scFv, or sdAb.         A molecule as claimed in claim 38, which comprises Fc.         The molecule of any one of claims 37 to 39, wherein the additional antibody is a humanized antibody or a human antibody comprising a human immunoglobulin constant region.         The molecule of any one of claims 37 to 38, wherein the additional antibody or an antigen-binding fragment of the antibody, and the antibody of any one of claims 1 to 27, 33 or the antigen-binding fragment of the antibody Combined with or without linkers.         The molecule of claim 41, wherein the carboxy terminus of the amino acid sequence possessed by the additional antibody or the antigen-binding fragment of the antibody is bound to a linker, and the carboxy terminus of the amino acid sequence possessed by the linker is For example, the antibody of any one of claims 1 to 27 and 33 or the antigen-binding fragment of the antibody is combined.         The molecule of claim 42, comprising: the carboxy terminus of the amino acid sequence possessed by the additional antibody or the antigen-binding fragment of the antibody is bound to a linker, and further the carboxyl group of the amino acid sequence possessed by the linker Amino acid sequences obtained by binding the ends to the following antibodies or antigen-binding fragments of the antibody: an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 19 or an antigen-binding fragment of the antibody, comprising An antibody having an amino acid residue at positions 2 to 243 of SEQ ID NO: 22 or an antigen-binding fragment of the antibody, or an antibody comprising an amino acid residue at positions 2 to 241 of SEQ ID NO: 25 or the antibody Antigen-binding fragment.         The molecule according to any one of claims 35 to 42, wherein in the antibody or the antigen-binding fragment of the antibody according to any one of claims 1 to 27, 33, the variable region is from the amine terminal side of the antibody Combined in the order of the heavy chain variable region and the light chain variable region, or combined in the order of the light chain variable region and the heavy chain variable region, optionally: i) having a linker between the two variable regions; ii) the amino-terminus of the variable region on the amino-terminus side has a glycine residue; iii) the carboxyl-terminus of the variable region on the carboxy-terminus side, a linker, a FLAG tag, and / or a His tag are bonded.         The molecule of claim 44 comprising the additional antibody or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 19, or an antigen-binding fragment of the antibody An antibody comprising an amino acid residue at positions 2 to 241 of sequence identification number 25 or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 60 or the An antigen-binding fragment of an antibody, an antibody comprising an amino acid residue at positions 2 to 241 of sequence identification number 64, or an antigen-binding fragment of the antibody, an amino acid residue comprising 2 to 243 of sequence identification number 66 An antibody of the residue or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 68 or an antigen-binding fragment of the antibody, the second to An antibody having an amino acid residue at position 243 or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 72 or an antigen-binding fragment of the antibody, including sequence recognition Resistance to amino acid residues 2 to 243 of No. 74 Or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of SEQ ID NO: 76 or an antigen-binding fragment of the antibody, an amine comprising 2 to 243 of sequence identification number 78 An antibody having an amino acid residue or an antigen-binding fragment of the antibody, an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 80 or an antigen-binding fragment of the antibody, an An antibody having an amino acid residue at positions 2 to 243 or an antigen-binding fragment of the antibody, or an antibody comprising an amino acid residue at positions 2 to 243 of sequence identification number 84 or an antigen-binding fragment of the antibody.         The molecule of any one of claims 36 to 45, wherein the additional antibody is an anti-cancer target antibody.         The molecule of any one of claims 36 to 46, which is bispecific.         The molecule of any one of claims 35 to 47, which is a polypeptide.         A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence possessed by a molecule as claimed in claim 48.         A vector comprising a polynucleotide as claimed in claim 49.         A cell that produces a polynucleotide as in claim 49 or a vector as in claim 50 or a molecule as in claim 48.         A method for producing a molecule that binds to human CD3 and cynomolgus CD3, comprising a step of culturing a cell as claimed in claim 51, and a step of recovering a molecule that binds to human CD3 from the culture.         A molecule that binds to human CD3 and cynomolgus monkey CD3, which is obtained by a method such as claim 52.         A pharmaceutical composition containing a molecule according to any one of claims 35 to 48 and 53 as an active ingredient.         The pharmaceutical composition according to claim 54, which induces cytotoxicity to a target cell by redirecting the T cell to the target cell.